Process, tube and device for the preparation of wound healant composition

ABSTRACT

The present invention is related to the field of tissue regeneration. It concerns more particularly new processes, tubes and devices for thrombin, platelet concentrate and wound healant preparations, alone or in combination with cell extracts, cell compositions and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/596,268, filed on Jan. 14, 2015, which is the divisional of U.S.application Ser. No. 13/634,020, filed Nov. 13, 2012. U.S. Pat. No.8,945,537, which is the U.S. national stage application of InternationalPatent Application No. PCT/IB2011/000684, filed Mar. 11, 2011, thedisclosures of which are hereby incorporated by reference in theirentirety, including all figures, tables and amino acid or nucleic acidsequences.

FIELD OF THE INVENTION

The present invention is related to the field of tissue regeneration. Itconcerns more particularly new processes, tubes and devices forthrombin, platelet concentrate and wound healant preparations,compositions and uses thereof.

BACKGROUND OF THE INVENTION

The importance of biological autologous materials in the healing processhas been well documented. Most importantly, two biological autologousmaterials have been shown to be directly implicated in the formation ofthe structure of blood clots, which provide a haemostatic barrier whoserole is to ensure hemostasis and seal the wound: (1) fibrin, whichderives from the separation of plasma fibrinogen into two strandsthrough the action of thrombin, and (2) the activated membranes ofplatelets. The wound healing process is generally presented as thesuccession of a coagulation phase, an inflammatory process and aregeneration process. The coagulation phase (blood clotting or clotformation) is a complex process whereby a damaged blood vessel wall iscovered by a fibrin clot to stop hemorrhage and the repair of thedamaged vessel is initiated by the release in large quantities ofcytokines and growth factors from platelet alpha granules. The formationof blood clots (formed in physiological conditions by fibrin, plateletsand red blood cells, among other blood components) is a naturalphenomenon that results from tissue trauma and its role in the woundhealing process, as well as in the union of bone fractures, iswell-known.

Blood coagulation is the result of the complex interaction of a numberof protein clotting factors through a cascade. In general, damage to thevascular endothelium exposes subendothelial structures, which attractplatelets and induce them to aggregate reversibly. The protein thrombin,formed during activation of the coagulation pathway generates insolublecross-linked fibrils of the protein fibrin and causes the platelets toaggregate irreversibly. The resulting platelet-fibrin clot is aneffective barrier against loss of blood from the vascular system andalso serves as a scaffold for subsequent repair of the lining of theblood vessel.

The inflammation process, which follows the formation of a blood clot,is stimulated by numerous vasoactive mediators and chemotactic factors(specific signals in the form of proteins) released by white blood cellsand platelets. These signals attract macrophages that “clean” the sitefrom bacteria and foreign particles as well as red blood cells beforethe migration of new cells. The tissue regeneration phase involves thechemoattraction and the mitosis of the undifferentiated cells in thescaffold (or growth matrix) formed by the blood clot. The new cellswhich multiply under the stimulation of platelet growth factors willreplace damaged or destroyed cells injured by macrophages. Growthfactors and numerous plasma proteins, also called signaling molecules,which promote cell migration and division within blood clots, play acrucial role in the wound healing process.

Bioadhesive sealants and fibrin glues represent a relatively newtechnological advance that duplicates the biological process of thefinal stage of blood coagulation. Clinical reports document the utilityof fibrin glue in a variety of surgical fields, such as, cardiovascular,thoracic, transplantation, head and neck, oral, gastrointestinal,orthopedic, neurosurgical, and plastic surgery. At the time of surgery,the two primary components comprising the fibrin glue, fibrinogen andthrombin, are mixed together to form a clot. The clot adheres to thenecessary tissues, bone, or nerve within seconds, but is then slowlyreabsorbed by the body in approximately 10 days by fibrinolysis.Important features of fibrin glue is its ability to: (1) achievehaemostasis at vascular anastomoses particularly in areas which aredifficult to approach with sutures or where suture placement presentsexcessive risk; (2) control bleeding from needle holes or arterial tearswhich cannot be controlled by suturing alone; and (3) obtain haemostasisin heparinized patients or those with coagulopathy. See, Borst, H. G.,et al., J. Thorac. Cardiovasc. Surg., 84:548-553 (1982); Walterbusch, G.J, et al., Thorac Cardiovasc. Surg., 30:234-235 (1982); and Wolner, F.J, et al., Thorac. Cardiovasc. Surg., 30:236-237 (1982).

Theoretically, it is possible to amplify the effects of these firstphases in the wound-healing cascade by discarding the red blood cellsand increasing the concentration of growth factors.

Blood clotting amplification can be defined as the formation of an“enriched clot (EC)”. ECs are obtained through the use of plateletconcentrates and have been described in Platelets and Megacaryocytes2004, vol 1 & 2, as “Structure and signals”, Ed. Gibbins andMahaut-Smith, Humana Press, New Jersey. Platelet-rich plasma (PRP) canbe defined as an autologous concentrate of platelets in a small volumeof plasma; it has been developed as an autologous biomaterial and hasproven to be useful in the healing and regeneration of tissues (Marx etal., 2004, J. Oral Maxillofac. Surg., 62, 489-496). PRP not onlyconsists in a platelet concentrate but also contains growth factors(such as platelet-derived growth factor: PDGF, vascular endothelialgrowth factor: VEGF, transforming growth factor: TGF and epidermalgrowth factor: EGF, etc.) that are actively secreted by platelets andare known to have a fundamental role in wound healing initiationprocess.

For example, PDGF is known to initiate connective tissue healing,including bone regeneration and repair. PDGF also increases mitogenesis(healing cells), angiogenesis (endothelial mitosis into functioningcapillaries) and macrophage activation. VEGF released by the leukocytesis also known to have potent angiogenic, mitogenic and vascularpermeability-enhancing activities on endothelial cells. TGF-[beta]promotes cell mitosis and differentiation for connective tissue andbone, acts on mesenchymal stem cells, preosteoblasts and fibroblasts andinhibits osteoclast formation. EGF is known to induce epithelialdevelopment and promote angiogenesis. Platelet concentrates aregenerally used in dental implantology and bone surgery, notably in theUSA. Various techniques of preparation of PRP by centrifugationprocesses have been developed. However, due to the sensitivity of theplatelet cells and the variability of the efficiency of the methods ofseparation of the platelets from the red blood cells, a greatvariability exists among the methods used for the preparation ofplatelet concentrates. The automated settings from Biomet PCCS & GPS(Marx et al., 2004, above), present the drawback of being a complexprocess with prohibitive costs for the process of a large blood sample.In those systems, there is also an important loss of valuable biologictissue from the patients, therefore there is the need for thedevelopment of a reliable process collecting the plasma cells with highyields, ease of use and cost effectiveness.

In addition, the obtaining of platelet concentrates still needs the useof relatively complex kits and costly dedicated machinery and theequally costly involvement of specialized technicians. This drawbackmakes the current known methods of preparation of PRP not adapted to apoint-of-care use.

Further, the preparation of cells in view of cellular or tissueregeneration for use in transplantation, post-operative regeneration orfor aesthetic purpose is faced to the long-term conservation problem ofcells and tissues. Tissue or cell cryoconservation is generally used forthe long-term maintaining of tissues or cells, notably platelets, butthis technique has shown serious drawbacks and problems such as crystalformation, osmotic problems, aggregation, inhibition of proteinsynthesis ability, stress protein expression in response to thermalstress. Therefore, tissue or cell cryoconservation is known to alter thecell viability and stability (Agence française de sécurité sanitaire,2003; Arnaud et al., 1999, Cryobiology, 38, 192-199; Tablin et al.,2001, Cryobiology, 43(2), 114-23). Some of the cryoconservation sideeffects may be limited by the use of anti-freezing agents such as DMSOor glycerol or other cryopreservatives (U.S. Pat. No. 5,891,617, Oh etal., Cornea, 26, 840-846) but the concentration of these agents has tobe adapted to limit their toxicity and side effects.

Therefore, there is a need for new or alternative method of preparationof cells and tissues suited for use extemporaneously while preservingtheir integrity, notably in terms of growth factors, secretion abilityand viability.

SUMMARY OF THE INVENTION

The invention relates to the field of tissue regeneration. It concernsmore particularly new processes, tubes and devices for thrombin,platelet concentrate and wound healant preparations, compositions anduses thereof. The invention also relates to new cell formulations, newplatelet-rich plasma (PRP) formulations, methods of preparation of newcell formulations or PRP formulations, use of such cell or PRPpreparations, optionally admixed with a cell extract, such as anautologous extract of keratinocytes, bone marrow cells, fibroblasts,periosteum or corneal cells, melanocytes and Langerhans cells; fatcells; fat tissue; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes; umbilical cord cells; mesenchymal stem cells(MSCs); preadipocytes; pre-endothelial cells; Schwann cells; or Achillestendon cells.

In one aspect, the invention provides a method for preparing acompletely autologous and extemporaneous wound healant or tissue healantcomposition. All of the blood components for the autologous woundhealant or tissue healant composition are derived from a single patientto whom the autologous wound healant or tissue healant composition willbe applied (same patient).

In one aspect, the invention provides a process or method for thepreparation of thrombin serum, comprising the steps of:

a) Collecting whole blood in a tube containing a thixotropic gel,

b) Centrifuging the tube until liberation of thrombin serum, and

c) Collecting the thrombin serum.

In another aspect, the invention provides a process or method for thepreparation of a wound healant composition or tissue healantcomposition, comprising the steps of:

a) Collecting whole blood preferably in a tube containing hyaluronicacid, a thixotropic gel and/or an anticoagulant preferably sodiumcitrate,

b) Centrifuging the tube preferably until migration of red blood cellsunder the thixotropic gel and preferably until migration of hyaluronicacid above the enriched plasma.

c) Optionally mixing the hyaluronic acid and the enriched plasma,preferably by inverting the tube, and

d) Collecting the supernatant containing hyaluronic acid and theenriched plasma.

In another aspect, the invention provides a tube for collecting andseparating a fluid sample comprising:

i) two distinct parts differing in size and diameter,

ii) a filter separating the two parts, and

iii) optionally a thixotropic gel and an anticoagulant.

In a preferred embodiment of the invention, the tube consists of thefeatures as illustrated in FIGS. 1 to 14.

In another aspect, the invention relates to a blood bag system or bloodcollection tubes device comprising multiple bags or tubes useful for thecollection, storage, use and delivery of blood components.

In a preferred aspect, the invention provides a blood bag system orblood collection tubes device according to FIG. 15.

In another aspect, the invention provides a blood bag system or bloodcollection tubes device comprising a single entry conduit connected to amultiple conduit adapter with adapter conduits connected to at least twobags or tubes, wherein each adapter conduit of the multiple conduitadapter is connected to one single bag or tube.

The blood collection tubes are preferably evacuated, sealed and filledwith thixotropic gel and anticoagulant.

DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification illustrate preferred embodiments of the presentinvention, and together with the description, serve to explain theprinciples of the invention.

FIGS. 1A and 1B are schematic representations of a tube according to theinvention.

FIGS. 2 and 3 are schematic representations of the interior of a tubeaccording to the invention.

FIG. 4 is a schematic representation of the interior of the filter of atube according to the invention. The filter comprises an internal layerwith funnels and an external layer with trapezoid-like structures.

FIG. 5A is a schematic representation of the interior of a tubeaccording to the invention.

FIG. 5B is a schematic representation of the cap and tube junction.

FIG. 6 is a schematic representation of the cap.

FIGS. 7A and 7B represent two distinct views of the filter of a tubeaccording to the invention.

FIG. 8 represents an underneath view of the filter of a tube accordingto the invention. FIG. 8 also represents the four symmetrical series of3 ranges of openings of the external layer or lower layer according tothe invention.

FIG. 9 represents an upper view of the filter of a tube according to theinvention.

FIG. 9 also represents the four symmetrical series of 2 ranges ofopenings of the internal layer or upper layer according to theinvention.

FIGS. 10 and 11 represent detailed views of the filter of a tubeaccording to the invention.

FIG. 12 represents a detailed view of the centric part of the filter ofa tube according to the invention.

FIG. 13 is a detailed representation of the interior of the filter of atube according to the invention. The filter comprises an internal layerwith funnels and an external layer with trapezoid-like structures. Eachfunnel and trapezoid is integrated into the filter in an alternatemanner (a first trapezoid is followed by a first funnel, followed by asecond trapezoid, followed by a second funnel and ending by a thirdtrapezoid).

FIG. 14 is a detailed view of the filter comprising funnels andtrapezoids of a tube according to the invention.

FIG. 15 is a view of the blood bag system or blood collection tubesdevice of the invention.

FIG. 16 is a schematic representation of the method for preparing awound or tissue healing composition comprising PRP and hyaluronic acid.

DETAILED DESCRIPTION OF THE INVENTION

The following paragraphs provide definitions of the terms according tothe invention and are intended to apply uniformly throughout thespecification and claims unless an otherwise expressly set outdefinition provides a broader definition.

The expression “thixotropic” means a gel that becomes more fluid as aresult of agitation or pressure, i.e., a gel which viscosity isdecreasing as a result of agitation or pressure. The term viscosityrefers to those characteristics of the specified material(s) determiningthe degree of gelation, such as for example the firmness or hardness ofthe material, the degree to which the material resists flowing like afluid. A thixotropic gel according to the invention comprising apolyester gel or a mixture thereof which is water insoluble andchemically inert to blood constituents which can be used in accordancewith the invention. Typical thixotropic gels are used in blood cellseparation for diagnostics and proteomics purposes. A thixotropic gel isalso herein referred to as a “cell selector gel”. Other gels may be usedin the present invention.

The expression “point-of-care” means all services provided to patientsat the bedside.

The expression “phlebotomy accessories” or “venipuncture accessories”means accessories that allow the puncture of a vein with a needle forthe purpose of drawing blood.

Alternative expressions for “wound healant” or “wound sealant” or“tissue healant” or “tissue sealant” or “wound healing composition” or“tissue healing composition” are “bioadhesive sealant” or “fibrin glue”.

The expression “wound healant” or “wound sealant” or “tissue healant” or“tissue sealant” or “wound healing composition” or “tissue healingcomposition” or “bioadhesive sealant” or “fibrin glue” means an agent ora composition that is able to promote and/or increase the speed and/orquality of cicatrisation of a wound. Wound healants or sealants are ableto promote tissue regeneration. The expression “wound” means any damagedtissue, for example following trauma or surgery. Wounds in mammalsinclude for example bed sores, ulcers, lacerations and burns, graftsites (graft donor and acceptor sites), fistulas, periodontal tissuedamages, diabetic non-healing wounds, consequences of traumas or anysurgery act. In its general sense the expression is intended to alsoencompass skin damages where the skin surface presents some depressionwithout necessarily a cut on its surface such as age-related tissuedamages (e.g., wrinkles) and scars such as for example acne (especiallyafter dermabrasion treatment) or rubella scars. The expression “PRP”means a platelet-rich plasma, preferably of mammal origin or humanorigin, more preferably autologous, prepared by the process of theinvention in order to pellet and remove erythrocytes and concentrate theplasma in leucocytes, thrombocytes and adhesion proteins as compared tonative whole blood. The expression “autologous” or “autogenic” or“autogenous” means an in-vivo method wherein a single donor's blood,tissue and/or cell is used and wherein the blood, tissue and/or cellextracted from this donor is intended for use on the same donor. Asopposed, “allogeneic” methods are using blood, tissue and/or cell fromone or more third parties for use on a donor (“homologous” or“heterologous”). An autologous product avoids some of the commonproblems associated with the use of biological materials from thirdparties, such as for example screening to assure that the donor wasbiologically or immunologically compatible with the patient andpotential contamination with hepatitis, HIV, prion, Creutzfeldt-Jakobdisease and the like. The expression “coagulation activator” means anagent, for example an enzyme, that is able to trigger or activatecoagulation of plasma and platelet aggregation. A coagulation activatorcomprises a thrombin activator and/or a fibrinogen activator and/orthrombin and/or an autologous thrombin and/or an autologous thrombinserum and/or calcium chloride and/or calcium gluconate. Coagulation maybe combined in order to change the stiffness of compositions.

The expression “thrombin activator” means an agent that is able toactivate thrombin and to trigger coagulation. Typical thrombinactivators are certain co factors such as sodium or calcium. Inpracticing this invention, thrombin activation preferably occurs in thepresence of calcium ions. Calcium ions are generally added to theplatelet concentrate as a salt solution to provide a final concentrationgenerally of or about 0.1 mg/mL of platelet concentrate. Suitablecalcium salts include, without limitation, CaCO3, CaSO4 or CaCl2. Apreferred calcium salt for use in the invention is calcium gluconate(CaGL). CaGL is available as calcium gel injection, USP 10% (Regen Lab,Switzerland). The expression “fibrinogen activator” means an agent thatis able to activate the conversion of fibrinogen into fibrin andtriggers the formation of the clot. Typical fibrinogen activators arethrombin or batroxobin. The term thrombin may include calcifiedthrombin, in particular, from or about 100 to about 10 units of thrombinper 1 mL of 10% of aqueous calcium gluconate solution; it may includecalcified bovine thrombin, allogeneic thrombin or recombinant humanthrombin, preferably autologous thrombin. A fibrinogen activator can bean enriched thrombin composition such as thrombin compositions asdescribed in U.S. Pat. No. 6,472,162 or an autologous thrombin serumaccording to the invention. The expression “therapeutically effectiveamount” means the amount or amounts of the constituent elements orcombination thereof necessary to enhance wound healing such as, forexample, the reduction in the volume or surface area of a wound, theincrease in the amount of granulation tissue or other biologicalmaterial facilitating collagen lay down, vascular in growth, fibroblastproliferation or overall healing. All of the versions of the inventiondescribed herein are assumed to have the therapeutically effectiveamount(s) of constituent substances, or combinations thereof. By theexpression “pharmaceutically acceptable carrier” is intendedpharmaceutically acceptable additional ingredients such as stabilizers,antimicrobial agents, buffers, adjuvants, anaesthetics, corticosteroidsand the like. By the expression “cosmetically acceptable carrier” isintended cosmetically acceptable additional ingredients such asstabilizers, buffers, colouring agents, flavouring agents, adjuvants,and the like.

The expression “Cyclic Olefin Copolymer” (COC) or “Cyclic OlefinPolymer” (COP) means an amorphous polymer, Ethylene Copolymer: COC: COP:Cyclo Olefinecopolymer: Cyclic Olefin Polymer; Ethylene-norborneneCopolymer. COPs use a single type of monomer whereas COCs use differenttypes of monomers. The invention encompasses cyclic olefin copolymersbased on different types of cyclic monomers and polymerization methods.The Cyclic olefin copolymers or polymers of the present invention may beproduced by chain copolymerization of cyclic monomers such as8,9,10-trinorborn-2-ene (norbornene) or1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene with ethene,Ticona's TOPAS, Mitsui Chemical's APEL, or by ring-opening metathesispolymerization of various cyclic monomers followed by hydrogenation (forexample Japan Synthetic Rubber's ARTON, Zeon Chemical's Zeonex andZeonor).

The expression “hyaluronic acid” (also called hyaluronan or hyaluronate)means an anionic, nonsulfated glycosaminoglycan distributed widelythroughout connective, epithelial, and neural tissues. It is uniqueamong glycosaminoglycans in that it is nonsulfated, forms in the plasmamembrane instead of the Golgi, and can be very large, with its molecularweight often reaching the million. One of the chief components of theextracellular matrix, hyaluronan contributes significantly to cellproliferation and migration.

The expression “chitosan” means a linear polysaccharide composed ofrandomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit)and N-acetyl-D-glucosamine (acetylated unit). Chitosan is producedcommercially by deacetylation of chitin, which is the structural elementin the exoskeleton of crustaceans (crabs, shrimp, etc.) and cell wallsof fungi. The degree of deacetylation (% DD) can be determined by NMRspectroscopy, and the % DD in commercial chitosans is in the range60-100%. On average, the molecular weight of commercially producedchitosan is between 3800 to 20,000 daltons. A common method for thesynthesis of chitosan is the deacetylation of chitin using sodiumhydroxide in excess as a reagent and water as a solvent. This reactionpathway, when allowed to go to completion (complete deacetylation)yields up to 98% product. The amino group in chitosan has a pKa value of˜6.5, which leads to a protonation in acidic to neutral solution with acharge density dependent on pH and the % DA-value. This makes chitosanwater soluble and a bioadhesive which readily binds to negativelycharged surfaces such as mucosal membranes. Chitosan enhances thetransport of polar drugs across epithelial surfaces, and isbiocompatible and biodegradable.

In one aspect, the invention provides a process or method for thepreparation of thrombin serum, comprising the steps of:

a) Collecting whole blood preferably in a tube preferably containing athixotropic gel,

b) Centrifuging the tube until liberation of thrombin serum, and

c) Collecting the thrombin serum.

The act of drawing blood initiates clotting reactions, and unlesssomething is done to stop the process, a clot will naturally form.

Preferably, the thixotropic gel is located near the bottom of the tube.During centrifugation, the red blood cells will migrate under the gel.Meanwhile, polymerization of fibrinogen occurs with the formation of aclot on the gel. Under sufficient centrifuge force and/or sufficientcentrifugation time, this clot will further precipitate forming a fibrinmesh which will liberate a liquid supernatant called serum comprisingenriched activated thrombin. Thrombin is an enzyme stimulatingcoagulation.

Advantageously, a thrombin serum according to the invention can beobtained in a few steps only by simply centrifuging a tube containingwhole blood and a thixotropic gel during sufficient centrifugation time.Advantageously, the method for the preparation of thrombin serumaccording to the invention provides a ready to use thrombin serum.

The thrombin serum is herein also referred to as thrombin enrichedpreparation, thrombin enriched serum, enriched activated thrombin serum,enriched activated thrombin preparation, thrombin-rich activated serum,thrombin-rich activated preparation.

In another aspect, the invention provides a process or method for thepreparation of thrombin serum, comprising the steps of:

a) Collecting whole blood preferably in a tube preferably containing athixotropic gel,

b) Centrifuging the tube until red blood cells migrate under thethixotropic gel and preferably until formation of a fibrin mesh on thethixotropic gel, and

c) Collecting the supernatant or thrombin serum.

In one preferred embodiment of the invention, the centrifugation step isperformed at a force of about 1500 g during approximately 30 minutes. Ina further embodiment, the centrifugation step is performed at forcebetween about 1000 g and up to about 2000 g for a time selected fromabout 20 min up to about 40 min, preferably at 1500 g for a timeselected from about 25 min up to about 35 min, preferably at 1500 g forabout 30 min.

Preferably, the centrifugation step is performed for a sufficient lengthof time until liberation of thrombin serum.

Advantageously, the methods of the present invention allow conservationof the liquid serum, the thrombin remaining soluble.

Standard methods consist in triturating the clot until liberation ofthrombin serum. Advantageously, this step is not needed in thepreparation of a thrombin serum according to the present invention. Thisalternative thrombin serum may also be used as a thrombin enrichedpreparation in the context of the invention.

Advantageously, no coagulation agent is used and, coagulation occursspontaneously. This has the advantages of saving costs and simplifyingthe process. As no citrate is present in the tube, advantageously nocoagulation agent (also referred to as restoring agent) is necessary toinitiate coagulation. Advantageously, no ethanol solution and/or calciumchloride is required.

Advantageously, the process or method for the preparation of thrombinserum herein described is simple to perform, necessitating a reducedamount of time of human presence as no clot trituration is required,representing an economic advantage over old methods of preparation.

An alternative autologous thrombin serum to be used as a thrombinenriched preparation in the context of the invention is prepared by anold process which comprises the addition to a patient's whole bloodsample (e.g., 10 mL) collected in a tube, a 95% v. ethanol solution(e.g., 1 mL) and calcium chloride 10% (e.g., 1 mL). The mixture is thenallowed to precipitate for about 30 min at room temperature. After 30min, almost 80% of the anti-thrombin (among other proteins likefibrinogen) is precipitated; then the tube is centrifuged at or about1500 g for about 8 to 10 min and the autologous thrombin serum is readyfor use in combination with a platelet-rich concentrate.

Preferably, the invention provides a process or method for thepreparation of autologous thrombin serum. Preferably, all the aspectsand/or embodiments of the present invention are for autologous use.Accordingly, this invention provides a method for preparing a completelyautologous thrombin serum wherein the donor and receiver is the sameperson or animal.

In one embodiment of the invention, the tube for the preparation ofthrombin serum is made of glass, preferably a glass separator tubecontaining a polyester-based thixotropic gel.

In a most preferred embodiment, the method for the preparation of thethrombin serum uses a tube according to the invention wherein no citrateis added.

In another aspect, the invention provides a process or method for thepreparation of a wound or tissue healing composition, comprising thesteps of:

a) Collecting whole blood preferably in a tube preferably containing athixotropic gel,

b) Centrifuging the tube preferably until liberation of thrombin serum,

c) Collecting the supernatant or thrombin serum, and

d) Admixing the thrombin serum with a PRP composition or an isolatedplatelet concentrate composition.

In another aspect, the invention provides a process or method for thepreparation of a wound or tissue healing composition, a cell compositionand/or a cell preparation comprising the steps of:

a) Collecting whole blood preferably in a tube preferably containing athixotropic gel,

b) Centrifuging the tube preferably until liberation of thrombin serum,

c) Collecting the thrombin serum,

d) Admixing the thrombin serum with a PRP composition or an isolatedplatelet concentrate composition, and

e) Admixing the resulting composition of step d) with a cell extract,cell composition, TCP, chitosan, hyaluronic acid, cream, cream mask, fatcells, fat tissue, bone marrow concentrate, lubricin, cd-gelatin,botulinum toxin and/or stem cells.

In one embodiment, the whole blood is collected into at least one tube.The tube may herein be referred to as a separator tube. Preferably, allthe methods and/or processes of the present invention may use one ormore tubes according to the invention.

In one embodiment, all the methods and/or processes of the presentinvention may use the whole blood collected in a blood bag system orblood collection tubes device according to the invention or a device orkit comprising such a blood bag system or blood collection tubes device.

An “empty” tube refers herein to a tube wherein no substances, nocompositions or else have been inserted and/or added into the tube.

Any process or method of the present invention may be prepared using atleast one tube according to the invention.

In another aspect, the invention provides a process or method for thepreparation of a wound healant composition or tissue healantcomposition, comprising the steps of:

a) Collecting whole blood in a tube,

b) Centrifuging the tube, and

c) Collecting the clot.

Advantageously, no coagulation agent is used for the preparation of thewound healant composition or tissue healant composition. Advantageously,the wound healant composition or tissue healant composition represents aready to use composition. Such composition may be directly applied ondiabetic ulcers.

Preferably, the process or method for the preparation of a wound healantcomposition or tissue healant composition is for use in dentistry and/ororthopedics.

In another aspect, the invention provides a wound healant composition ortissue healant composition comprising a clot for use in dentistry,orthopedics, arthritis, pseudo-arthritis or else. In one embodiment, thewound healant composition or tissue healant composition comprising theclot is applied in a dental cavity, on a diabetic ulcer, perforatingulcer, diabetic perforating ulcer, or else. Method of treatment and useof the healant composition or tissue healant composition comprising aclot is also encompassed by the invention.

In one embodiment, the wound healant composition or tissue healantcomposition may be combined with tricalcium phosphate (TCP) or with anybone substitute preferably before the formation of the clot.

The wound healant composition comprising TCP may preferably be preparedin a vial.

In one embodiment, the wound healant composition or tissue healantcomposition may be combined with hyaluronic acid (HA) preferably beforethe formation of the clot.

In another aspect, the present invention provides a method or processfor the preparation of a platelet concentrate composition orplatelet-rich plasma composition, comprising the steps of:

a) Centrifuging whole blood in a tube according to the inventionpreferably comprising sodium citrate and/or a thixotropic gel, and

b) Collecting the platelet-rich plasma.

The centrifugation step will eliminate the Red Blood Cells (RBCs) fromthe plasma. The top phase is a platelet rich plasma (PRP), and thebottom phase is anticoagulated whole blood minus the platelet richplasma.

Preferably, the centrifugation step is performed at a force of or about1500 g up to about 2000 g. Preferably, the centrifugation step isperformed for a sufficient length of time to form a barrier between theplasma containing the platelets, the lymphocytes and the monocytes andthe gel containing the erythrocytes.

Preferably, the separation step b) is made by collecting the supernatantfrom the top of said barrier. In one embodiment, the enriched plateletrich plasma is separated from the full plasma by removing half of thesupernatant containing the platelet poor plasma. Preferably, theenriched plasma is enriched in leucocytes, thrombocytes and adhesionproteins (for example, fibronectin (soluble protein) or vitronectin(protein secreted by platelets)) as compared to native whole blood.

In one embodiment, the tube used for the preparation of platelet richplasma is selected from:

i) a glass separator tube containing a polyester-based thixotropic geland a buffered sodium citrate solution at 0.10 M,

ii) a polyethylene terephthalate separator tube containing a highlythixotropic gel formed by a polymer mixture and an anhydrous sodiumcitrate at 3.5 mg/mL,

iii) a Cyclic Olefin Copolymer (COC) or Cyclic Olefin Polymer (COP)separator tube containing a polyester-based thixotropic gel and abuffered sodium citrate solution at 0.10 M, or

iv) a Cyclic Olefin Copolymer (COC) or Cyclic Olefin Polymer (COP)filter separator tube containing a buffered sodium citrate solution at0.10 M or an anhydrous sodium citrate at 3.5 mg/mL.

In a most preferred embodiment, the method for the preparation of aplatelet concentrate uses a tube according to the invention with theaddition of citrate (for example a buffered sodium citrate solution at0.10 M or an anhydrous sodium citrate at 3.5 mg/mL).

In another aspect, the present invention provides a method or processfor the preparation of a wound healant or tissue healant composition,comprising the steps of

a) Centrifuging whole blood in a tube according to the inventionpreferably comprising sodium citrate and/or a thixotropic gel,

b) Collecting the platelet-rich plasma, and

c) Admixing the platelet-rich plasma with a cell extract, cellcomposition, TCP, chitosan, hyaluronic acid, cream, cream mask, fatcells, fat tissue, bone marrow concentrate, lubricin, cd-gelatin,botulinum toxin and/or stem cells.

In another aspect, the present invention provides a wound healant ortissue healant composition comprising:

a) a platelet-rich plasma or plasma concentrate according to theinvention and

b) TCP, chitosan, hvaluronic acid, cream, cream mask, fat cells, fattissue, bone marrow concentrate, lubricin, cd-gelatin, botulinum toxinand/or stem cells.

In another aspect, the present invention provides a wound healant ortissue healant composition comprising:

a) a thrombin serum according to the invention,

b) a platelet-rich plasma or plasma concentrate preferably according tothe invention, and

c) TCP, chitosan, hvaluronic acid, cream, cream mask, fat cells, fattissue, bone marrow concentrate, lubricin, cd-gelatin, botulinum toxinand/or stem cells.

The formation of a clot is a multi-step process or cascade and severalof these steps require the presence of calcium ions. By removing thecalcium ions present in whole blood, as is the effect when the blood iscollected in citrate, the blood can be prevented from clotting. Acalcium chelating agent is a chemical that reacts with the calcium,present in blood, in such a fashion that the calcium can no longerfunction in blood coagulation. The most common chelating agent is a saltof citric acid (citrate), since it has the fewest side effects on thecomponents of the clotting system. By collecting blood into a mediumcontaining a calcium chelating agent such as citrate, sample collectionand further preparations of the citrated sample can be performed over atime period of up to several hours. Preferred calcium chelating agent issodium citrate.

While a 3.5 mg/ml sodium citrate collection medium is that which isfrequently used to collect and preserve blood, the person skilled inthis art will recognize that the ratio of sodium citrate to whole bloodcould be in a different range.

In another aspect, the present invention provides an isolated plateletconcentrate composition comprising:

a) plasma:

b) platelets at a concentration of at least 300×10⁹ cells/L;

c) white blood cells at a concentration of at least 7×10⁹ cells/L;

d) fibrinogen at a concentration of at least 3 mg/L;

and wherein the erythrocyte concentration is less than 0.6×10′² cells/L.

A platelet rich plasma composition is also referred to herein as plasmaconcentrate composition.

In one embodiment, a platelet concentrate composition or platelet richplasma composition according to the invention may be combined withtricalcium phosphate (TCP) and/or any one substitute for use as volumecorrector (TCP at 10-30 microns), in dentistry, orthopedics (TCP at 50microns).

In another aspect, the present invention provides a method or processfor the preparation of a wound healant composition or tissue healantcomposition, comprising the steps of:

a) Centrifuging whole blood in a tube comprising hyaluronic acid,

b) Optionally separating the enriched platelet rich plasma andhyaluronic acid from the full plasma, and

c) Optionally mixing the enriched platelet rich plasma and hyaluronicacid.

Preferably, the tube comprises hyaluronic acid, preferably at the bottomof the tube, followed by a thixotropic gel and then an anticoagulant,preferably sodium citrate (FIG. 16).

Preferably, the separation step b) is made by collecting the supernatantcontaining the enriched platelet rich plasma and hyaluronic acid fromthe top of said barrier. In one embodiment, the enriched platelet richplasma and hyaluronic acid is separated from the full plasma by removinghalf of the supernatant containing the platelet poor plasma. Preferably,the enriched plasma is enriched in leucocytes, thrombocytes and adhesionproteins (for example, fibronectin (soluble protein) or vitronectin(protein secreted by platelets)) as compared to native whole blood.

In another aspect, the invention provides a process or method for thepreparation of a wound healant composition or tissue healantcomposition, comprising the steps of:

a) Collecting whole blood preferably in a tube containing hyaluronicacid, a thixotropic gel and/or an anticoagulant preferably sodiumcitrate,

b) Centrifuging the tube preferably until migration of red blood cellsunder the thixotropic gel and preferably until migration of hyaluronicacid above the enriched plasma,

c) Optionally mixing the hyaluronic acid and the enriched plasma,preferably by inverting the tube,

d) Collecting the supernatant containing hyaluronic acid and theenriched plasma, and

e) Optionally further mixing said hyaluronic acid and said enrichedplasma.

In another aspect, the invention provides a process or method for thepreparation of a wound healant composition or tissue healantcomposition, comprising the steps of:

a) Collecting whole blood preferably in a tube containing hyaluronicacid, a thixotropic gel and/or an anticoagulant preferably sodiumcitrate.

b) Centrifuging the tube preferably until formation of a hyaluronic acidlayer as first layer from the top of the tube followed by a second layerconsisting of enriched plasma or PRP,

c) Optionally mixing the hyaluronic acid and the enriched plasma,preferably by inverting the tube,

d) Collecting the supernatant containing hyaluronic acid and theenriched plasma, and

e) Optionally further mixing said hyaluronic acid and said enrichedplasma.

The centrifugation step will eliminate the Red Blood Cells (RBCs) fromthe plasma. After centrifugation, the top phase is hyaluronic acid withunderneath a platelet rich plasma (PRP), followed by the thixotropic geland the bottom phase is anticoagulated whole blood containing the redblood cells minus the platelet rich plasma (FIG. 16). Duringcentrifugation, hyaluronic acid migrates above plasma (FIG. 16).

Preferably, the tube contains about 1 ml to about 2 ml of hyaluronicacid, about 2 g of cell selector or thixotropic gel and about 1 ml ofsodium citrate at 0.109M.

A schematic representation of the method for the preparation of a woundhealant composition comprising hyaluronic acid and PRP is provided inFIG. 16.

Preferably, the enriched platelet rich plasma and hyaluronic acid aremixed by simple inversion of the tube.

Advantageously, when mixing the enriched platelet rich plasma andhyaluronic acid, the hyaluronic acid expands itself inflated by plasmaand cells.

Advantageously, the wound healant composition or tissue healantcomposition obtained is a ready to use composition. Advantageously, thewound healant composition or tissue healant composition obtained is aviscous gel or biological glue suitable for injection, and for examplemay be used as mechanical support or filler.

Advantageously, the method or process for the preparation of a woundhealant composition, tissue healant composition or viscous gel iseconomical, simple and rapid.

Preferably, the centrifugation step is performed for a sufficient lengthof time till migration of red blood cells under the thixotropic gel andpreferably until migration of hyaluronic acid above the enriched plasma.

In one preferred embodiment of the invention, the centrifugation step isperformed at a force of about 1500 g during approximately 5 minutes. Ina further embodiment, the centrifugation step is performed at forcebetween about 1000 g and up to about 2000 g for a time selected fromabout 3 min up to about 7 min, preferably at 1500 g for a time selectedfrom about 3 min up to about 7 min.

In another aspect, the invention provides a tube comprising hyaluronicacid and an anticoagulant. In another aspect of the invention, theinvention provides a tube comprising hyaluronic acid, an anticoagulantand whole blood. Preferably, the anticoagulant is sodium citrate.

In another aspect, the invention provides a tube comprising hyaluronicacid and a cell selector gel. In another aspect, the invention providesa tube comprising hyaluronic acid, a cell selector gel and whole blood.Preferably, the cell selector gel is a thixotropic gel.

In another aspect, the invention provides a tube comprising hyaluronicacid, an anticoagulant and a cell selector gel. In another aspect, theinvention provides a tube comprising hyaluronic acid, an anticoagulant,a cell selector gel and whole blood. Preferably, the anticoagulant issodium citrate. Preferably, the cell selector gel is a thixotropic gel.Preferably, hyaluronic acid is located at the bottom of the tube,followed by a thixotropic gel and above an anticoagulant, preferablysodium citrate (FIG. 16).

In another aspect, the invention provides a tube comprising hyaluronicacid and PRP. In another aspect, the invention provides a tubecomprising hyaluronic acid, PRP and a cell selector gel. In anotheraspect, the invention provides a tube comprising hyaluronic acid, PRPand an anticoagulant. In another aspect, the invention provides a tubecomprising hyaluronic acid, PRP, an anticoagulant and a cell selectorgel. Preferably, the anticoagulant is sodium citrate. Preferably, thecell selector gel is a thixotropic gel.

Preferably, a tube according to the invention is used in a method orprocess according to the invention.

In another aspect, the invention provides a composition comprisinghyaluronic acid and PRP. In another aspect, the invention provides acomposition comprising hyaluronic acid and PRP, wherein the compositionis obtained by a method for the preparation of a wound healantcomposition or tissue healant composition according to the invention.

In another aspect, the invention provides a kit or medical devicecomprising a tube according to the invention.

In further embodiments, the invention provides a tube, which may be usedfor the preparation of a wound healant composition or tissue healantcomposition, selected from:

i) a glass separator tube containing a polyester-based thixotropic gel,a buffered sodium citrate solution at 0.10 M and hyaluronic acid,

ii) a polyethylene terephthalate separator tube containing a highlythixotropic gel formed by a polymer mixture, an anhydrous sodium citrateat 3.5 mg/m and hyaluronic acid,

iii) a Cyclic Olefin Copolymer (COC) or Cyclic Olefin Polymer (COP)separator tube containing a polyester-based thixotropic gel, a bufferedsodium citrate solution at 0.10 M and hyaluronic acid, or

iv) a Cyclic Olefin Copolymer (COC) or Cyclic Olefin Polymer (COP)filter separator tube containing hyaluronic acid and a buffered sodiumcitrate solution at 0.10 M or an anhydrous sodium citrate at 3.5 mg/mL.

In further preferred embodiments, the invention provides a tubeaccording to the invention, which may be used for the preparation of awound healant composition or tissue healant composition, furthercomprising citrate and hyaluronic acid (for example hyaluronic acid anda buffered sodium citrate solution at 0.10 M or an anhydrous sodiumcitrate at 3.5 mgmL).

Preferably, no phthalates are used for human use.

Alternatively, hirudin, benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide(BAPA), heparin, citrate, acid citrate dextrose (ACD),citrate-theophylline-adenosine-dipyridamole (CTAD) orpotassium-ethylenediaminetetra-acid (EDTA) may be used asanticoagulants.

In another aspect, the present invention provides a wound healantcomposition or tissue healant composition comprising:

a) plasma;

b) platelets at a concentration of at least 300×10⁹ cells/L:

c) white blood cells at a concentration of at least 7×10⁹ cells/L:

d) fibrinogen at a concentration of at least 3 mg/L:

e) about 1 ml to about 2 ml of hyaluronic acid;

and wherein the erythrocyte concentration is less than 0.6×10¹² cells/L.

In one embodiment, a wound healant composition or tissue healantcomposition according to the invention may be combined with tricalciumphosphate (TCP) and/or any one substitute for use as volume corrector(TCP at 10-30 microns), in dentistry, orthopedics (TCP at 50 microns).

In one embodiment, a wound healant composition, a tissue healantcomposition, a haemostatic agent, a platelet concentrate composition, ora platelet rich plasma composition according to the invention may becombined in the blood collection tube with hvaluronic acid.

In another aspect, the invention provides a process or method for thepreparation of a haemostatic agent, a wound healant composition ortissue healant composition, comprising the step of mixing a thrombinserum or thrombin serum according to the invention with a platelet richplasma composition, wound healant composition or tissue healantcomposition according to the invention.

In another aspect, the present invention provides a process or methodfor the preparation of a wound healant composition, tissue healantcomposition or haemostatic agent comprising:

a) Providing a platelet concentrate or platelet concentrate of theinvention.

b) Admixing the platelet concentrate with a coagulation activator,thrombin serum or thrombin serum according to the invention, and

c) Optionally admixing a cell extract, such as extract of keratinocytes,bone marrow, fibroblasts, periosteum or corneal cells, melanocytes andLangerhans cells; fat cells; muscle cells such as imoblasts andsatellite cells; osteoblasts; chondrocytes; blood progenitor cells,umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells. Schwann cells or Achilles tendon cells.

Preferably, an autologous platelet concentrate, an autologous thrombinand/or an autologous cell extract is/are used. More preferably, anautologous platelet concentrate and an autologous thrombin and anautologous cell extract are used. Preferably, tubes according to theinvention are used.

In another aspect, the present invention provides a process or methodfor the preparation of a wound healant composition or tissue healantcomposition or haemostatic agent comprising:

a) Admixing a platelet concentrate with an autologous thrombin serumaccording to any aspects of the invention, and

b) Optionally admixing at least one autologous cell extract, such asextract of keratinocytes, bone marrow, fibroblasts, periosteum orcorneal cells, melanocytes and Langerhans cells; fat cells; muscle cellssuch as myoblasts and satellite cells; osteoblasts; chondrocytes;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells or Achilles tendon cells.

In another aspect, the present invention provides a process or methodfor the preparation of a wound healant composition or tissue healantcomposition or haemostatic agent comprising:

a) Admixing an autologous platelet concentrate with a thrombin serum,wherein the autologous platelet concentrate and/or the thrombin serumare prepared using tubes according to the invention, and

b) Optionally admixing at least one autologous cell extract, such asextract of keratinocytes, bone marrow, fibroblasts, periosteum orcorneal cells, melanocytes and Langerhans cells; fat cells; muscle cellssuch as myoblasts and satellite cells; osteoblasts; chondrocytes;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells or Achilles tendon cells.

In another aspect, the present invention provides a process or methodfor the preparation of a wound healant composition or tissue healantcomposition or haemostatic agent comprising:

a) Providing a platelet concentrate, preferably an autologous plateletconcentrate according to the invention,

b) Admixing the platelet concentrate with a thrombin serum, preferablyan autologous thrombin according to the invention, and

c) Optionally admixing at least one cell extract, preferably anautologous cell extract, such as extract of keratinocytes, bone marrow,fibroblasts, periosteum or corneal cells, melanocytes and Langerhanscells; fat cells; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes; umbilical cord cells; mesenchymal stem cells(MSCs), preadipocytes, pre-endothelial cells, Schwann cells or Achillestendon cells.

Preferably, the invention provides a method or process for thepreparation of an autologous wound healant composition or tissue healantcomposition or haemostatic agent.

An autologous wound healant composition or tissue healant composition orhaemostatic agent herein means a composition wherein either the plateletconcentrate or thrombin serum is autologous.

In a most preferred aspect, the invention provides a method or processfor the preparation of a completely autologous wound healant compositionor tissue healant composition or haemostatic agent. A completelyautologous wound healant composition or tissue healant composition orhaemostatic agent herein means a composition wherein both the plateletconcentrate or thrombin serum are autologous. In this preferred aspectof the invention, all of the blood components for the wound healantcomposition or tissue healant composition or haemostatic agent arederived from the same patient or animal to whom the wound healantcomposition or tissue healant composition or haemostatic agent will beapplied.

In another aspect, the invention provides a method or process for thepreparation of a completely autologous wound healant composition ortissue healant composition or haemostatic agent in combination with atleast one autologous cell extract. In this aspect, the plateletconcentrate, the thrombin serum and the cell extract(s) are allautologous and are therefore all derived from the same patient oranimal.

In another aspect, the present invention provides a process or methodfor the preparation of an autologous wound healant composition orautologous tissue healant composition or autologous haemostatic agentcomprising:

a) Mixing an autologous platelet concentrate, preferably an autologousplatelet concentrate according to the invention, with an autologousthrombin serum, preferably an autologous thrombin according to theinvention, and

b) Optionally admixing at least one cell extract, such as extract ofkeratinocytes, bone marrow, fibroblasts, periosteum or corneal cells,melanocytes and Langerhans cells; fat cells; muscle cells such asmyoblasts and satellite cells; osteoblasts; chondrocytes; umbilical cordcells; mesenchymal stem cells (MSCs), preadipocytes, pre-endothelialcells, Schwann cells or Achilles tendon cells,

wherein the autologous platelet concentrate and the autologous thrombinare both derived from the same patient or same animal.

In another aspect, the present invention provides a process or methodfor the preparation of an autologous wound healant composition orautologous tissue healant composition or autologous haemostatic agentcomprising:

a) Mixing an autologous platelet concentrate, preferably an autologousplatelet concentrate according to the invention, with an autologousthrombin serum, preferably an autologous thrombin according to theinvention, and

b) Optionally admixing at least one autologous cell extract, such asextract of keratinocytes, bone marrow, fibroblasts, periosteum orcorneal cells, melanocytes and Langerhans cells; fat cells; muscle cellssuch as myoblasts and satellite cells; osteoblasts; chondrocytes;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells or Achilles tendon cells,

wherein the autologous platelet concentrate, the autologous thrombin andthe autologous cell extract are all derived from the same patient orsame animal.

The platelet rich plasma, autologous platelet rich plasma, thrombinserum, autologous thrombin serum, wound healant composition or tissuehealant composition or haemostatic agent of the present invention may becombined with one or more cell extracts. In one embodiment, the cellextract is selected from keratinocytes, bone marrow, fibroblasts,periosteum or corneal cells, melanocytes and Langerhans cells; fatcells; muscle cells such as myoblasts and satellite cells; osteoblasts:chondrocytes; umbilical cord cells; mesenchymal stem cells (MSCs),preadipocytes, pre-endothelial cells, Schwann cells or Achilles tendoncells.

A wound healant composition or tissue healant composition or haemostaticagent of the present invention will stimulate cellular regeneration,acting as a biological glue in order to promote tissue adhesion.

In one embodiment, instead of thrombin serum, an alternative coagulationactivator may be used such as calcium chloride, preferably calciumgluconate.

In one embodiment, multiple coagulation activators may be used incombination, preferably thrombin serum with calcium gluconate.

Preferably, the coagulation activator or thrombin serum is admixed withthe platelet concentrate in a vol. ratio (plateletconcentrate:coagulation activator) of about 10:1 up to about 10:3.

In a preferred embodiment of the invention, the liquid serum iscollected in a 1:1 proportion, 3:1 proportion or a 10:1 proportion tothe platelet rich plasma. The specific proportion used will alter thehaemostatic agent stiffness. In a 3:1 proportion, a strong haemostaticagent is obtained. With a 10:1 proportion, a softer agent is obtained.

In one embodiment, the PRP or plasma concentrate alone or in combinationwith a cell extract, as well as preparations or compositions of thepresent invention may be combined or integrated with a soak acellularmatrix to be applied directly on a wound, or may be cultivated inlaboratory before application. A colagene matrix or synthetic matrix maybe used, for example the Integra matrix.

Another embodiment contemplates mixing human recombinant thromboplastindirectly with a platelet rich plasma to form a wound healant compositionor tissue healant composition or haemostatic agent. Alternatively, humanrecombinant thromboplastin is utilized to generate thrombin in a smallaliquot of plasma and then the resulting thrombin is combined with theplatelet rich plasma to form a wound healant composition or tissuehealant composition or haemostatic agent.

In one embodiment, the tubes used may have either wettable surfaces(such as, silica, diatomaceous earth, kaolin, etc.) or non-wettablesurfaces (such as plastic, siliconized glass, etc.). Since surfaces playa role in activating blood coagulation, the surface of the separatortube chosen is dependent on whether clot formation is desired quickly orslowly. Chemical activators, such as kaolin, can also be used to speedup the clotting time; however, their subsequent removal would also benecessary.

Advantageously, the preparation of platelet rich plasma, plateletconcentrate, thrombin serum, wound healant composition or tissue healantcomposition or haemostatic agent of the present invention do notnecessitate the presence of ethanol and/or calcium. By using autologousthrombin according to the invention, the present invention doesn'tnecessitate restoring the clot-forming process. As such, no agent (orrestoration agent) such as calcium chloride or calcium gluconate isrequired to reverse the effects of the anticoagulation agent (inrestoring the coagulation activity of citrated blood).

Although calcium chloride is the well-known calcium salt for use asrestoration agent, any calcium salt which functions in a similar mannerto calcium chloride may be considered as restoration agent. Similarly,although many blood coagulation reactions are currently believed torequire calcium ions as cofactors, any substance that is known orsubsequently found to be functionally equivalent to calcium infacilitating these coagulation reactions may be considered asrestoration agent, either individually or in combination with calcium.If the anticoagulation agent used was heparin, then heparinase would beused as restoration agent to reverse the effect of the anticoagulationagent.

Advantageously, the invention provides a platelet rich plasma, plateletconcentrate, thrombin serum, wound healant composition or tissue healantcomposition or haemostatic agent wherein the risks associated with theuse of bovine and recombinant human thrombin are eliminated.

Advantageously, a higher concentration of PRP, growth factors,leukocytes, fibrinogen and/or other proteins is obtained with themethods of the invention.

Advantageously, a 2 fold concentration of PRP, growth factors,leukocytes, fibrinogen and/or other proteins is obtained in comparisonwith normal hematological levels.

Advantageously, the methods of the present invention confer an optimumcellular productivity for cellular expansion.

Advantageously, the methods of the present invention permit manipulationof the blood in an entirely closed circuit during the entire process,from blood collection, manipulation until application or injection tothe patient. All the devices and kits are therefore adapted for anentirely closed circuit manipulation in order to avoid direct contact ofthe blood with air.

Advantageously, the methods of the present invention reduce oxidativestress and reduce manipulation time ex-vivo.

Advantageously, the methods of the present invention permit theformation of a solid autologous suturable membrane composed ofaggregated platelets and activated polymerized fibrinogen.

The preparations of the present invention (for example bone marrow cellpreparation) may be used alone or then admixed to the plateletconcentrate according to the invention or centrifuged again with calciumgluconate to form a suturable membrane and applied or injected with anapplicator to the injured site of the patients.

A bone marrow cell preparation according to the invention is useful forthe treatment of bone defect or cartilage defect. The bone cellpreparation may be used alone or in combination with a plasmaconcentrate according to the invention. A cartilage membrane may be usedas well with calcium gluconate.

In one preferred embodiment of the invention, the centrifugation step(e.g., for the suturable membrane) is performed at a force of about 3000g during 15 to 25 minutes. In one embodiment, the centrifugation step isperformed at a force between about 2500 g and up to about 3500 g for atime selected from about 10 min up to about 30 min.

In one embodiment of the invention, the whole blood is collected from ahuman being or animal. A preferred embodiment of the invention is tocollect the whole blood from a human being.

In another aspect, the present invention provides a wound or tissuehealant composition or haemostatic agent prepared according to theinvention comprising:

a) plasma;

b) platelets at a concentration of at least 300×10⁹ cells/L;

c) white blood cells at a concentration of at least 7×10⁹ cells/L;

d) fibrinogen at a concentration of at least 3 mg/L;

e) coagulation activator, autologous thrombin serum or autologousthrombin serum according to the invention,

f) optionally an autologous cell extract, such as an extract ofkeratinocytes, bone marrow cells, osteoblasts; chondrocytes,fibroblasts, periosteum or corneal cells, melanocytes and Langerhanscells; fat cells; muscle cells such as myoblasts and satellite cells;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells, tendon cells or pancreas isletcells; and wherein the erythrocyte concentration is less than 0.6×10¹²cells/L.

In another aspect, the present invention provides a wound or tissuehealant composition or haemostatic agent comprising:

a) plasma;

b) platelets at a concentration of at least 300×10⁹ cells/L;

c) white blood cells at a concentration of at least 7×10⁹ cells/L;

d) fibrinogen at a concentration of at least 3 mg/L;

e) autologous thrombin serum according to the invention;

f) optionally an autologous cell extract, such as an extract ofkeratinocytes, bone marrow cells, osteoblasts; chondrocytes,fibroblasts, periosteum or corneal cells, melanocytes and Langerhanscells; fat cells; muscle cells such as myoblasts and satellite cells;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells, tendon cells or pancreas isletcells; and wherein the erythrocyte concentration is less than 0.6×10¹²cells/L.

Preferably, the coagulation activator or autologous thrombin serum is ina vol. ratio (platelet concentrate:coagulation activator) of about 10:1to about 10:3.

Advantageously, the plasma of the present invention represents an idealcell culture medium over known ones. Advantageously, the plasma of thepresent invention represents an ideal medium for the transport of cells.Accordingly, the plasma of the present invention contains all the cellsand growth factors for an optimum cell growth and survival.

Accordingly, the plasma of the present invention represents a verysuitable medium for the implantation of cells to a patient, for exampleas an atopic application like surgical wounds, for anti-age injections,intra-articular injections, intra-muscular injections, or for pancreasregeneration (pancreatic islets).

In another aspect, the present invention provides a device for thepreparation of a platelet concentrate composition or platelet richplasma composition or haemostatic agent or wound or tissue healantcomposition or autologous thrombin serum according to the invention.

In another aspect, the present invention provides a device comprising atleast one tube according to the invention.

Preferably, the device has an inlet for introducing said whole blood, isheld in a vacuum intended to aspirate the whole blood sample, issterile, has a usable vacuum of or about 8 to about 10 mL and issuitable for undergoing centrifugation.

In another aspect, the present invention provides a use of a plateletconcentrate composition or platelet rich plasma composition orhaemostatic agent or wound or tissue healant composition or thrombinserum according to the invention for the manufacture of a medicament forhealing of wounds or for promoting bone or periodontum growth and/orbone and/or tissue regeneration.

In another aspect, the present invention provides a use of plateletconcentrate composition or platelet rich plasma composition orhaemostatic agent or wound or tissue healant composition or thrombinserum according to the invention for the manufacture of a cosmeticpreparation for use as anti-aging agent or skin repairing agent such asa scar repairing agent, a wrinkle filling and/or repairing agent.

In another aspect, the invention provides a platelet concentratecomposition or platelet rich plasma composition or haemostatic agent orwound healant composition or tissue healant composition or thrombinserum according to the invention for use as cosmetic preparation,esthetic preparation, aging management, volume corrector, wrinklefeeling, brown spot reduction and/or hair stimulator. In one embodiment,the platelet concentrate composition or platelet rich plasma compositionor haemostatic agent or wound healant composition or tissue healantcomposition according to the invention is applied on and/or around theeyes, lips, eyelids, face, neck, chest, scalp, hair, hands and all therest of the body and/or male and female genitalia.

In another aspect, the invention provides a platelet concentratecomposition or platelet rich plasma composition or haemostatic agent orwound healant composition or tissue healant composition or thrombinserum according to the invention for use in ligament and/or cartilagereconstitution. Advantageously, ligament and/or cartilage reconstitutiontime using a composition of the present invention is divided by a factor2 or 3 in comparison with known methods.

In one embodiment, the cosmetic preparation and/or esthetic preparationis combined with a cosmetic agent, cosmetic cream or cosmetic mask.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a platelet concentrate composition or plateletrich plasma composition or haemostatic agent or wound or tissue healantcomposition or thrombin serum according to the invention and apharmaceutically acceptable carrier.

In another aspect, the present invention provides a cosmetic compositioncomprising a platelet concentrate composition or platelet rich plasmacomposition or haemostatic agent or wound or tissue healant compositionor thrombin serum according to the invention and a cosmeticallyacceptable carrier.

In another aspect, the present invention provides an implantable devicefor use in tissue regeneration therapy comprising:

a) a permeable core comprising a platelet concentrate composition orplatelet rich plasma composition or haemostatic agent or wound healantcomposition according to the invention optionally in combination withhyaluronic acid, and

b) optionally an external jacket surrounding said core, said jacketcomprising a biocompatible material, preferably bioresorbable,preferably hyaluronic acid.

In another aspect, the invention provides a kit comprising at least onetube according to the invention.

In another aspect, the invention provides a kit comprising a tube forthe preparation of a platelet concentrate according to the inventionand/or a tube for the preparation of thrombin serum according to theinvention.

In another aspect, the invention provides a kit comprising at least onetube for the preparation of a platelet concentrate composition orplatelet rich plasma composition or haemostatic agent or wound or tissuehealant composition or thrombin serum according to the invention.

In one embodiment, the kit further comprises one or more tubes for thepreparation of a platelet concentrate composition or platelet richplasma composition or haemostatic agent or wound or tissue healantcomposition or thrombin serum according to the invention.

In one embodiment, the kit further comprises phlebotomy accessories forthe preparation of the wound or tissue healant and an applicator device(e.g., a double syringe) for the simultaneous dispensation onto thewound of the platelet concentrate composition or platelet rich plasmacomposition or haemostatic agent or wound or tissue healant compositionor thrombin serum according to the invention.

In one embodiment, the kit is adapted for tissue regeneration.

In another aspect, the invention provides a method for promoting woundhealing or tissue healing and/or sealing and/or tissue and/or boneregeneration in a wound or tissue of a human or animal comprising:

a) Providing a wound healant or tissue healant composition orhaemostatic agent according to the invention, and

b) Applying a therapeutically effective amount of the wound or tissuehealant or haemostatic agent to a wound, a damaged tissue or a damagedbone.

In another aspect, the invention provides a method of treatmentcomprising:

a) Providing a wound healant or tissue healant composition orhaemostatic agent according to the invention, and

b) Applying a therapeutically effective amount of the wound or tissuehealant or haemostatic agent to a wound, a damaged tissue or a damagedbone.

In another aspect, the invention provides a method for inducingperiodontal regeneration in a wound or a periodontal defect of a mammalwith periodontal disease or other condition requiring periodontalregeneration comprising:

a) Providing a wound or tissue healant or haemostatic agent according tothe invention,

b) Applying a therapeutically effective amount of the wound or tissuehealant composition or haemostatic agent to the wound or tissue or theperiodontal defect or cavity,

c) Optionally inserting a periodontal barrier, and

d) Closing the wound or tissue.

Preferably, the barrier is positioned between the gingival tissue andthe wound or tissue treated according to steps a) and b). In oneembodiment, the barrier is selected from a membrane, a biodegradablepolymer and/or a biocompatible porous material.

The membrane may be obtained by combining PRP and calcium gluconate 10%in a proportion of 30 to 70 or 50 to 50 respectively. Centrifugation isperformed during approximately 30 minutes.

In another aspect, the invention provides a method for promoting skinregeneration in a scar or a wrinkle from human or animal comprising:

a) Providing a wound or tissue healant composition or haemostatic agentaccording to the invention, and

b) Filling the skin scar or wrinkle line with the said wound or tissuehealant composition or haemostatic agent.

In another aspect, the present invention provides a process for thepreparation of a cell composition, comprising the steps of:

a) Centrifuging whole blood in a tube according to the invention,

b) Optionally separating the enriched platelet rich plasma from the fullplasma,

c) Re-suspending the enriched plasma, and

d) Providing a cell extract such as an extract of dermal cells such askeratinocytes, fibroblasts, melanocytes and Langerhans cells; fat cells;bone marrow cells; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes; periosteal filter cells; corneal cells;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells, tendon cells or pancreas isletcells, and

e) Admixing the platelet concentrate obtained under step c) with thecell extract obtained in d).

Preferably, the centrifugation step is performed at a force of or about1500 g up to about 2000 g. Preferably, the centrifugation step isperformed for a sufficient length of time to form a barrier between theplasma containing the platelets, the lymphocytes and the monocytes andthe pellet containing the erythrocytes.

Preferably, the separation step b) is made by collecting the supernatantfrom the top of said barrier. In one embodiment, the enriched plateletrich plasma is separated from the full plasma by removing half of thesupernatant containing the platelet poor plasma. Preferably, theenriched plasma is enriched in leucocytes, thrombocytes and adhesionproteins (for example, fibronectin) as compared to native whole blood.

In another aspect, the present invention provides a process for thepreparation of a wound or tissue healing composition, comprising thesteps of:

a) Centrifuging whole blood in a tube or in a separator tube accordingto the invention,

b) Optionally separating the enriched platelet rich plasma from the fullplasma.

c) Re-suspending the enriched plasma, and

d) Admixing the platelet concentrate obtained under step c) with acoagulation activator, thrombin serum, autologous thrombin serum orautologous thrombin serum according to the invention,

e) Providing a cell extract such as an extract of dermal cells such askeratinocytes, fibroblasts, melanocytes and Langerhans cells; fat cells;bone marrow cells; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes: periosteal filter cells; corneal cells;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells, tendon cells or pancreas isletcells; and

f) Admixing the platelet concentrate admixture obtained under step d)with the cell extract obtained in e).

Preferably, the centrifugation step is performed at a force of or about1500 g up to about 2000 g. Preferably, the centrifugation step isperformed for a sufficient length of time to form a barrier between theplasma containing the platelets, the lymphocytes and the monocytes andthe pellet containing the erythrocytes.

Preferably, the separation step b) is made by collecting the supernatantfrom the top of said barrier. In one embodiment, the enriched plateletrich plasma is separated from the full plasma by removing half of thesupernatant containing the platelet poor plasma. Preferably, theenriched plasma is enriched in leucocytes, thrombocytes and adhesionproteins (for example, fibronectin) as compared to native whole blood.

Preferably, the coagulation activator or autologous thrombin serum is ina vol. ratio (platelet concentrate:coagulation activator) of about 10:1to about 10:3.

In another aspect, the present invention provides an isolated cellcomposition prepared according to the invention comprising:

a) plasma,

b) platelets at a concentration of at least 300×10⁹ cells/L,

c) white blood cells at a concentration of at least 7×10⁹ cells/L,

d) fibrinogen at a concentration of at least 3 mg/L, and

e) a cell extract, such as an extract of dermal cells such askeratinocytes, fibroblasts, melanocytes and Langherans cells; fat cells;bone marrow cells; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes; periosteal filter cells; corneal cells;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells, tendon cells or pancreas isletcells.

Preferably, the cells are at a concentration of about 10⁵ to about 10⁶cells/ml of plasma or enriched plasma and the erythrocyte concentrationis less than 0.6×10¹² cells/L.

In another aspect, the present invention provides an isolated cellcomposition prepared according to the invention comprising:

a) plasma,

b) platelets at a concentration of at least 300×10⁹ cells/L,

c) white blood cells at a concentration of at least 7×10⁹ cells/L,

d) fibrinogen at a concentration of at least 3 mg/L,

e) a coagulation activator, thrombin serum, autologous thrombin serum orautologous thrombin serum according to the invention, and

f) a cell extract, such as an extract of dermal cells such askeratinocytes, fibroblasts, melanocytes and Langerhans cells; fat cells;bone marrow cells; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes; periosteal filter cells; corneal cells;umbilical cord cells; mesenchymal stem cells (MSCs), preadipocytes,pre-endothelial cells, Schwann cells, tendon cells or pancreas isletcells.

Preferably, the coagulation activator or autologous thrombin serum is ina vol. ratio (platelet concentrate:coagulation activator) of about 10:1to about 10:3.

Preferably, the cells are at a concentration of about 10⁵ to about 10⁶cells/ml of plasma or enriched plasma and the erythrocyte concentrationis less than 0.6×10¹² cells/L.

In another aspect, the invention provides a wound or tissue healingcomposition or haemostatic agent comprising an isolated cell compositionaccording to the invention.

In another aspect, the invention provides a method for promoting woundor tissue healing and/or sealing and/or regeneration of a tissue and/ora cartilage and/or a bone and/or a nerve in a human or an animalcomprising:

a) Providing a wound or tissue healing composition, a haemostatic agentor a cell composition according to the invention, and

b) Applying a therapeutically effective amount of the wound or tissuehealing composition, haemostatic agent or cell composition to a wound, adamaged tissue or a damaged cartilage or a damaged bone.

In another aspect, the invention provides a method for increasingadipose tissue volume in a mammal with a dermal fat graft or othercondition requiring adipose tissue regeneration comprising:

a) Providing a fat cell composition according to the invention,

b) Applying a therapeutically or cosmetically effective amount of thefat cell composition in combination with PRP or plasma concentrateaccording to the invention to the dermal fat graft or the adipose tissuerequiring adipose tissue regeneration, and

c) Optionally inserting a surgical flap or implant, wherein the surgicalflap or implant is positioned in the site requiring regeneration orvolumetric amplification and the said surgical flap or implant comprisesa combination of a fat cell preparation according to the invention andPRP, plasma concentrate or enriched plasma material.

In another aspect, the invention provides a method for inducingmyocardial regeneration in a mammal with myocardial deficiency or othercondition requiring myocardial tissue regeneration comprising:

a) Providing a muscle cell or a bone marrow cell composition accordingto the invention, and

b) Applying a therapeutically effective amount of the muscle cellcomposition in combination with PRP or plasma concentrate according tothe invention to the myocardial tissue requiring regeneration.

In another aspect, the invention provides a method for inducing cornealregeneration in a mammal with corneal deficiency or other conditionrequiring corneal regeneration comprising:

a) Providing a corneal cell composition according to the invention, and

b) Applying a therapeutically effective amount of the corneal cellcomposition in combination with PRP or plasma concentrate according tothe invention to the corneal tissue requiring regeneration.

In another aspect, the invention provides a method for inducingarticular or cartilage regeneration in a mammal with articular orcartilage deficiency or other condition requiring articular or cartilagetissue regeneration comprising:

a) Providing a chondrocyte cell or bone marrow cell compositionaccording to the invention,

b) Applying a therapeutically effective amount of the chondrocyte cellcomposition in combination with PRP or plasma concentrate according tothe invention to the articular or cartilage tissue requiringregeneration, and

c) Optionally inserting a surgical flap or implant, wherein the surgicalflap or implant is positioned in the defect of the cartilage or under aperiosteal patch, and the surgical flap or implant comprises acombination of a chondrocyte or bone marrow cell composition accordingto the invention and PRP, plasma concentrate or enriched plasmamaterial.

In another aspect, the invention provides a method for promoting skinregeneration in a scar, a wrinkle or a fat deficiency from human orlower animal comprising:

a) Providing a wound or tissue healing composition, a haemostatic agentor a cell composition in combination with PRP or plasma concentrateaccording to the invention, and

b) Filling the skin scar, wrinkle line or fat deficiency with the woundor tissue healing composition, a haemostatic agent or a cell compositionin combination with PRP or plasma concentrate according to theinvention.

In another aspect, the invention provides a method for inducingperipheral nerve regeneration in a mammal with peripheral nerve damage,nerve suture or spinal cord injury or other condition requiringperipheral nerve regeneration comprising:

a) Providing a Schwann cell composition in combination with PRP orplasma concentrate according to the invention, and

b) Applying a therapeutically effective amount of the Schwann cellcomposition in combination with PRP or plasma concentrate to theperipheral nerve requiring regeneration.

In another aspect, the invention provides a method for inducing boneregeneration in a mammal with bone damage, bone deficiency or othercondition requiring bone regeneration comprising:

a) Providing a bone marrow cell or osteoblast cell composition incombination with PRP or plasma concentrate according to the invention,and

b) Applying a therapeutically effective amount of the bone marrow cellor osteoblast cell composition in combination with PRP or plasmaconcentrate according to the invention to the bone requiringregeneration.

In another aspect, the invention provides a method for the treatment oftype I diabetes, insulin-dependent diabetes or hyperglycaemia in amammal comprising:

a) Providing a pancreas islet cell composition in combination with PRPor plasma concentrate according to the invention, and

b) Applying a therapeutically effective amount of the pancreas isletcell composition in combination with PRP or plasma concentrate accordingto the invention to the patient, for example by injection.

In another aspect, the invention provides a method for the treatment ofurinary incontinence in a mammal or other condition requiring bladderregeneration comprising:

a) Providing a myoblast cell composition in combination with PRP orplasma concentrate according to the invention, and

b) Applying a therapeutically effective amount of the myoblast cellcomposition in combination with PRP or plasma concentrate according tothe invention to the bladder neck requiring regeneration.

In another aspect, the invention provides a method for the treatment ofanal incontinence in a mammal or other condition requiring anal muscleregeneration comprising:

a) Providing a myoblast cell composition in combination with PRP orplasma concentrate according to the invention, and

b) Applying a therapeutically effective amount of the myoblast cellcomposition in combination with PRP or plasma concentrate according tothe invention to the para-anal area requiring regeneration.

In another aspect, the invention provides a method for the treatment ofreflux oesophagitis or gastro-oesophageal reflux disorders in a mammalor other condition requiring oesophageal sphincter regenerationcomprising:

a) Providing a myoblast cell composition in combination with PRP orplasma concentrate according to the invention, and

b) Applying a therapeutically effective amount of the myoblast cellcomposition in combination with PRP or plasma concentrate according tothe invention to the oesophageal sphincter requiring regeneration.

In another aspect, the present invention provides a use of a wound ortissue healing composition, a haemostatic agent, a cell composition or acell preparation according to the invention for the manufacture of amedicament for healing of wounds or tissues, mesotherapy, intramuscular,or for promoting bone or periodontum growth and/or bone and/or tissueregeneration such as skin, cartilage, muscle, tendon, adipose tissue,cornea, peripheral nerves, spine or bone regeneration.

In another aspect, the present invention provides a use of a wound ortissue healing composition, a haemostatic agent, a cell composition or acell preparation according to the invention for the manufacture of acosmetic preparation for use as anti-aging agent or skin repairing agentsuch as scar repairing agent, lipoatrophy repairing agent or wrinklefilling and/or repairing agent.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a wound or tissue healing composition, ahaemostatic agent, a cell composition or a cell preparation according tothe invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a cosmetic compositioncomprising a wound or tissue healing composition, a haemostatic agent, acell composition or a cell preparation according to the invention and acosmetically acceptable carrier.

In another aspect, the present invention provides an implantable devicefor use in tissue regeneration therapy comprising:

a) a permeable core comprising a wound or tissue healing composition, ahaemostatic agent, a cell composition or a cell preparation of theinvention optionally in combination with hyaluronic acid, and

b) an external jacket surrounding said core, said jacket comprising abiocompatible material, preferably bioresorbable, preferably hyaluronicacid.

In one embodiment, the compositions, wound or tissue healantcompositions, cell extracts, cell compositions, plasma concentrate andthrombin preparations of the invention use allogeneic substances,compositions, blood or cells.

The uses, methods and compositions according to the invention are usefulin the regeneration and/or rejuvenation of tissues, bones and/orcartilages. The uses, methods and compositions according to theinvention are particularly useful in the treatment of diabeticneuropathic ulcers or decubitus sores: bone and cartilage damages suchas deep joint cartilage or chondral damages such as surgical repair oftom tendons; arthritis in joint caused by traumas or by aging: rotatorcuff disorders; non-healing wounds such as vasculitis induced wounds,for example in lower equine limb; periodontal diseases; implant surgery;cardiovascular, thoracic, transplantation, head and neck, oral,gastrointestinal, orthopedic, neurosurgical, and plastic surgery:mesotherapy and/or mesotherapy injections; cardiac muscle damages suchas in chronic cardiac failure, heart failure, ischemic and non-ischemicdisorders, cardiomyopathy: gastro-oesophageal reflux disease; anal orurinary incontinence; facial surgery such as facial surgery inducedalopecia (alopecia due to hair follicle loss in the side burn areas),hair loss, alopecia, face-lift surgery (rhytidectomy), rhinoplasty,dermal fat grafts (in the treatment of facial augmentation, congenitalhemiatrophy of the face such as congenital cartilage nose atrophy andlipoatrophy such as in HIV/AIDS suffering patients, genital dysfunction,erosion and arthroscopy); wound healing complications such as aftereyelid blepharoplasty; corneal disorders such as corneal opacity such asthose caused by chemical burns, affliction by Stevens-Johnson syndromeand corneal ulcers; scarring of the comea; dry eye syndrome;haematological diseases such as Thalassaemia: peripheral nerve damage,nerve suture and spinal cord injury; bone defects or disorders such asbone graft or bone fracture, skin damages or disorders such as acne(especially after dermabrasion treatment), burns, rubella or small poxscars, vitiligo, lipoatrophy, Kaposi's sarcoma, skin keloids orDupuytren's palmar fibromatosis.

The uses, methods and compositions according to the invention are usefulin tissue healing, including bone regeneration and repair, mitogenesis,angiogenesis and/or macrophage activation.

Additional advantages and novel features of this invention shall be setforth in part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theobjects and advantages of the invention may be realized and attained bymeans of the instrumentalities, combinations, compositions, and methodsparticularly pointed out in the appended claims.

The compositions, uses and methods according to the invention areparticularly useful in haemostasis, in the regeneration, revitalization,hydration and/or stimulation of tissue, as biological glue, bioadhesivesealant or biological filler.

Advantageously, the strong biological glue of the present invention hasa higher mechanical strength than other known ones, notably for allinvasive surgical interventions. Such biological glues of the presentinvention have an increased capacity to cure damaged tissue bycontaining appropriate cells, platelets, leukocytes, growth factors andother factors reducing/preventing eventual infections.

The compositions, uses and methods according to the invention areparticularly useful in wound care, surgery, injections for orthopedicand injections for esthetic, cosmetic or volume corrections.

In another aspect, the uses, methods and compositions according to theinvention are useful in the regeneration and/or rejuvenation of skintissues, particularly in promoting and/or initiating skin regenerationsuch as reducing skin wrinkles, deep wrinkles, acne (especially afterdermabrasion treatment), burns, rubella or small pox scars, vitiligo andlipoatrophy (e.g., anti-aging compositions and skin regenerationcompositions), amelioration of nasolabial lines and treatment of skindamages or disorders such as skin burns, Kaposi's sarcoma, skin keloidsor Dupuytren's palmar fibromatosis, in the reduction of pain associatedwith skin and tissue regeneration, for hemorrhoidal cushion, erectiledysfunction, caverna, cavernosal fibrosis, Peyronie's disease, vaginaand/or labia.

The compositions, uses and methods according to the invention areparticularly useful in wound or tissue healing, regeneration treatmentsor sports medicine for the knee, elbow, (tom) muscles, spine, spinaldisc, tendon, ligament, the treatment of traumatic or surgical woundssuch as the fitting and/or holding and/or sealing of native orprosthetic grafts (especially skin, bone grafts and/or dental prosthesesor implants or the like, including also the graft donor site); treatmentof arthritis, gonarthritis, tendinitis, rotator cuff, treatment ofvasculitis; ulcers such as diabetic neuropathic ulcers or decubitussores: radiodermatitis (e.g., after irradiation on an epidermoidal skincarcinoma) and closing fistulas (such as for cyclists).

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of cardiac disorders, cardiacregeneration such as in the treatment of heart failure, chronic cardiacfailure, ischemic and non-ischemic cardiac failure and cardiomyopathy.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of urinary and/or analincontinence.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of reflux oesophagitis and/orgastro-oesophageal reflux disorder.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of skin damages such as inskins damaged by radiation (radiodermatitis or sun damaged skin), agedskins or burned skins and/or in the amelioration of facial wrinkles,rhytids, acne (especially after dermabrasion treatment), burns, rubellaor small pox scars, vitiligo, lipoatrophy or lipodystrophy, Kaposi'ssarcoma, skin keloids or Dupuytren's palmar fibromatosis and/or in skinrejuvenation treatments.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of lipoatrophy such as inHIV/AIDS patients and in other congenital hemiatrophy of the face suchas congenital cartilage nose atrophy. Further, the compositions, usesand methods according to the invention are particularly useful in thetreatment of bone, cartilage and articular disorders such as chondraldamage, arthritis, cartilage and/or bone injury such as deep cartilagedamage and/or erosion and/or arthroscopy, tendon torn and rotator cuffin shoulder.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of hematological diseases suchas Thalassaemia.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of corneal disorders such asdry eye syndrome; corneal opacity such as those caused by chemicalburns, affliction by Stevens-Johnson syndrome; scarring of the corneaand corneal ulcers.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of peripheral nerve damage,nerve suture and spinal cord injury.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of type I diabetes,insulin-dependent diabetes and/or hyperglycaemia.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of bone defects or disorderssuch as bone graft or bone fracture.

The use of the resulting composition of the invention can be furthermodified before application and according to the therapeutic objective.

Compositions of the invention can be used together with bone fillingmaterials, especially resorbable filling materials such ashydroxyapatite (calcium phosphate ceramic used as a biomaterial) ordemineralised bone, or used as a mixture with bone extracts in a processfor the regrowth of bone for example in craniofacial and orthopaedicprocedures.

Depending on the use or disease, compositions of the invention can beused together with hyaluronic acid 10%, hyaluronic acid 20%, hyaluronicacid 30%, hyaluronic acid 40%, and/or hvaluronic acid 50%.

Compositions of the invention may be used as a wound sealant in plasticsurgery including burn grafting and other free skin graft applications,for example in oncology for favouring tissue regeneration, includingspeeding (neo)vascularization. The compositions according to theinvention are particularly useful in wound healing treatments at theskin graft donor site. The removal of a skin graft on a healthy skincreates a new wound at the donor's site which normally healsspontaneously between 12 to 14 days. However, this cicatrisation isextremely demanding for the body, especially if the donor site is broador the person is less resistant (e.g., burn victims, people sufferingfrom multiple traumas, people treated with corticoids, children orelderly) and the energetic losses are even increased by the loss inminerals, trace elements and proteins induced by the fluid losses fromthe new wound. In addition, important pain during the first 8 days isoften present on the graft donor's site. Pain reduction treatments areoften used such as the use of analgesics (e.g., morphine) and/orhydrocellular wound dressings, however pain remains present, especiallyduring the dressing change that occurs imperatively within 48 hours upto 1 week after the graft removal. In addition, the hydrocellular wounddressings have the drawbacks not only to be rather expensive but also bymaintaining humidity on the wound, to prevent its drying, to increasethe wound deepness, to favour the outbreak of bacterial infections andto lead to non-esthetic scars. Therefore, a stimulation of the skingraft donor site healing is very desirable.

Compositions of the invention are particularly adapted to chronic woundsthat may lack sufficient blood circulation to facilitate the woundhealing cascade.

The compositions and methods according to the invention may be also usedin the treatment of periodontal disease where a loss and/or a damage ofthe periodontal tissues is observed, such a treatment comprising forexample placing at the periodontal site or cavity in a human or a loweranimal in need of periodontal tissue regeneration a compositionaccording to the invention.

The compositions according to this invention are effective ineliminating or greatly reducing post-operative bleeding andextravasation or loss of serous or other fluid in these applications, inreducing the infection risk caused by most bacteria and/or enhancesconnective tissue formation compared to natural healing (i.e., noexogenous agents added) or to healing obtained through the use of otherplatelet concentrates prepared with known methods. The compositionsaccording to the invention are particularly useful in the preparation ofpharmaceuticals for promoting and/or initiating wound healing and/ortissue regeneration or for the preparation of cosmetic compositions forskin regeneration such as reducing skin wrinkles, acne (especially afterdermabrasion treatment), rubella or small pox scars, vitiligo andlipoatrophy (e.g., anti-aging compositions and skin regenerationcompositions).

The compositions of the present invention may be administered locally orinjected in the wound or in or near to the grafted organ or injectedsubcutaneously. Local administration may be by injection at the site ofinjury or defect or by insertion or attachment of a solid carrier at thesite, or by admixture with a cream or emulsion, or by inclusion in atissue or paper or hydrogel carrier, or by direct, topical applicationof the composition of the invention such as in the form of eye drops.Preferably, the compositions are readily syringable compositions. Themode of administration, the dosage administered, as single or multipledoses, to an individual will vary depending upon a variety of factors,including pharmacokinetic properties, patient conditions andcharacteristics (sex, age, body weight, health, size), extent ofsymptoms, concurrent treatments, frequency of treatment and the effectdesired.

The compositions of the present invention may be administered incombination with a co-agent useful in the treatment of tissueregeneration such as a healing agent, a wrinkle filler, an anti-agingagent such as an anti-aging vitamin complex, an antibacterial agent,antibiotic agent, a corticosteroid agent, an antalgic and analgesicagent, or an anesthetic agent like adrenaline, etc. The inventioncomprises compositions combined with a co-agent useful in the treatmentof tissue regeneration for simultaneous, separate or sequential use intissue regeneration therapy such as wound healing, bone and periodontumgrowth repair.

The compositions of the invention, the device and procedures for thepreparation of autologous platelet concentrates or cell compositions ofthe invention are particularly useful for therapeutic use, particularlyas autogenous biological glue in a haemostatic system intended toaccelerate the physiological process of tissue regeneration, for examplein dental implantology, skin and bone surgery, cartilage and tendonsurgery, corneal and peripheral nerve regeneration and cardiac surgery.The compositions of the invention, the device and procedures for thepreparation of autologous platelet concentrates and cell composition ofthe invention are particularly useful for cosmetic use, particularly asautogenous rejuvenation material intended to be used for example aswrinkle, scar or fat deficiency filler, alone or in combination with atleast one anti-aging agent.

The platelet concentrate of the invention may be combined with anautologous cell extract preparation such as for example keratinocytes,bone marrow cells, osteoblasts, chondrocytes, fibroblasts, periosteum,melanocytes and Langerhans cells; fat cells; bone marrow cells; musclecells such as myoblasts and satellite cells; periosteal filter cells;mesenchymal stem cells (MSCs), preadipocytes, pre-endothelial cells,corneal cells; umbilical cord cells; tendon cells or pancreatic isletcells. Keratinocytes can be harvested through a method described byReinwald and Green, 1975, Cell, 6(3):331-43. Other mentioned cells canbe harvested through methods described in “Culture de cellules animales;méthologies-applications”, 2003, Ed. Barlovatz-Meimom and Adolphe,INSERM editions, Paris. Alternatively, cell extracts are derived from acell bank or a cell culture or harvested as described in the Examplesbelow.

The platelet concentrate and cell compositions of the invention haveproven to be really beneficial in the acceleration and/or promotion ofthe healing process of wounds, even chronic unhealing wounds, leading tosuccessful closures where weeks of conventional therapies had failed andachieving a decrease in infection risks, an improvement in patient'srecovery and comfort, a reduction of medical care costs and a betteresthetic final result.

The compositions of the invention can of course be also prepared fromplasma derived from several identified donors. The invention is notlimited to autologous biological materials, such as collection ofconcentrated platelets from the wounded's own biological material. Theinvention encompasses the use of biological materials obtained from oneor more third parties, who need not be of the same species as thepatient whose wound is being treated with the wound healant compositiondescribed herein unless bio-incompatibility would result from the use ofsuch third party biological materials. In one embodiment, the inventionprovides a process for the preparation of a platelet concentratecomposition or a cell composition as described herein.

In another embodiment, the present invention provides a device for thepreparation of a platelet concentrate composition from whole blood asdescribed herein.

In a further embodiment, the invention provides a process for thepreparation of a thrombin serum and/or platelet concentrate compositionwherein the centrifugation step is performed at about 1500 g and up toabout 1700 g for a time selected from about 3 min up to about 15 min,preferentially at 1500 g for about 8 min.

In another further embodiment, the invention provides a process for thepreparation of a platelet concentrate composition wherein the tube hasan inlet for introducing said whole blood, is held in a vacuum intendedto aspirate the whole blood sample, is sterile, has a usable vacuum of 8to 10 mL and is suitable for undergoing centrifugation.

In another further embodiment, the invention provides a process for thepreparation of a platelet concentrate composition wherein the tube is apolyethylene terephthalate tube containing a highly thixotropic gelformed by a polymer mixture and an anhydrous sodium citrate at 3.5mg/mL.

In another embodiment, the present invention provides an isolatedplatelet concentrate composition obtainable from the process accordingto the invention.

In another embodiment, the invention provides an isolated plateletconcentrate composition obtained by a process according to the inventioncomprising:

a) plasma,

b) platelets at a concentration of at least 300×10⁹ cells/L, preferablyof at least 350×10⁹ cells/L, more preferably of at least 400×10⁹cells/L,

c) white blood cells at a concentration of at least 7×10⁹ cells/L,preferably of at least 8×10⁹ cells/L, and

d) fibrinogen at a concentration of at least 3 mg/L.

Preferably, the erythrocyte concentration is less than 0.4×10¹² cells/Lor 0.5×10¹² cells/L.

In another embodiment, the present invention provides a wound or tissuehealant composition comprising:

a) plasma,

b) platelets at a concentration of at least 300×10⁹ cells/L, preferablyof at least 350×10⁹ cells/L, more preferably of at least 400×10⁹cells/L,

c) white blood cells at a concentration of at least 7×10⁹ cells/L,preferably of at least 8×10⁹ cells/L.

d) fibrinogen at a concentration of at least 3 mg/L,

e) a thrombin serum according to the invention, and

f) optionally an autologous cell extract, such as extract ofkeratinocytes, bone marrow cells, fibroblasts, periosteum, melanocytesand Langerhans cells; fat cells; bone marrow cells; muscle cells such asmyoblasts and satellite cells; osteoblasts: chondrocytes; mesenchymalstem cells (MSCs), preadipocytes, pre-endothelial cells, periostealfilter cells; corneal cells; umbilical cord cells; tendon cells orpancreatic islet cells.

Preferably, the coagulation activator or thrombin serum is in a vol.ratio (platelet concentrate:coagulation activator) of about 10:1 toabout 10:3.

Preferably, the cells are at a concentration of about 10⁵ to about 10⁶cells/ml of plasma or enriched plasma and the erythrocyte concentrationis less than 0.4×10¹² cells/L or 0.5×10¹² cells/L.

In another embodiment, the invention provides a process for thepreparation of a wound or tissue healant composition as describedherein.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healant composition wherein thecoagulation activator which is admixed is calcium gluconate or 10%calcium chloride.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healant composition wherein thecoagulation activator which is admixed with a platelet concentrate (PRP)is a thrombin enriched preparation, which may further be combined withcalcium gluconate or calcium chloride. A method for preparing thrombinfor use in a biological glue is described in U.S. Pat. No. 6,472,162 bythe addition of 8 to 20% ETOH to a volume of plasma and this preparationmay be used as a thrombin enriched preparation in the context of theinvention. Alternatively, an autologous thrombin serum (ATS) can be usedas thrombin enriched preparation in the context of the invention. Analternative autologous thrombin serum according to the invention isobtained by a process comprising (i) the addition to a patient's wholeblood sample (e.g., 8 mL) collected in a separator tube of theinvention, a 10% of final volume of calcium chloride 10% (e.g., 1 mL)and a 10% of the final volume of a preparation of 95% v. ethanolsolution (e.g., 1 mL) and (ii) precipitation for about 30 min at roomtemperature. After 30 min, a centrifugation at or about 1500 g for about8 to 10 min is performed. In a further preferred embodiment, thethrombin enriched preparation and preferably the autologous thrombinserum according to the invention is admixed with a platelet concentrate(PRP) directly on the wound.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healant composition according to theinvention further comprising a step b′) wherein the activatedplatelet-rich preparation composition (obtained by the admixing of theplatelet concentrate with the coagulation activator or autologousthrombin serum) obtained in step b) may be partially dehydrated by thecontact of a wound dressing covered by a soft hydrophobic layer to avoidcontamination with micro-strings from the dressing in order to obtain asemi-solid gel that can be manipulated by appropriate instruments, forexample to fill a cavity or tissue deficiency, or as a growth matrix(“scaffold”) while waiting for the reconstitution of the autogenousextracellular matrix. The obtained wound or tissue healant isparticularly useful in a method for inducing periodontal regeneration ina wound, a tissue or a periodontal defect or a cavity.

In another further embodiment, the platelet concentrate, wound or tissuehealant composition, or cell compositions according to the invention maybe combined with a hydrogel like the Albugel (EP1543846) preparation of100% Albumin or any other hydrogel resulting from the reticulation ofAlbumin and other chemical compound like polyethylene glycol or anyother ingredient, using a paper based highly hydrophilic carrier toleave in contact with the skin until the platelet rich plasma isabsorbed.

The tensile strength of the wound or tissue healant compositions of thepresent invention can be affected by the addition of calcium ions.Consequently, if a stronger wound or tissue healant composition isdesired more calcium ions may be added at the time the serum is mixedwith the platelet concentrate. Alternatively, calcium ions may beintroduced directly into the platelet concentrate, and the wound ortissue healant compositions, respectively, will form.

As discussed in further detail below, the time period necessary for theformation of the wound or tissue healant compositions of the presentinvention is dependent on the quantity of serum added. A 1:4, 1:2 and3:4 ratio of serum to platelet concentrate results in the formation ofthe wound or tissue healant compositions in approximately 90, 55 and 30seconds, respectively. Furthermore, thrombin is preferably used withinfive hours of preparation, preferably within two hours and ideallyimmediately. As thrombin is active at room temperature still after 10days, thrombin may be used at a later stage. Alternatively, the serumcan be chilled or frozen indefinitely, preferably used before 1 month ofstorage.

The wound or tissue healant compositions of this invention may be usedfor sealing a surgical wound by applying to the wound a suitable amountplatelet concentrate once it has begun to gel. Moreover, due to the factthat the wound or tissue healant compositions of the present inventionmay be prepared solely from blood components derived from the patientthat is to receive the wound or tissue healant compositions there is azero probability of introducing a new blood transmitted disease to thepatient.

The methods of the present invention may be further modified so that theformed wound or tissue healant composition functions not only as ahaemostatic agent, but also as an adjunct to wound healing and as amatrix for delivery of drugs and proteins with other biologicactivities. For example, it is well known that fibrin glue has a greataffinity to bind bone fragments, which is useful in bone reconstruction,as in plastic surgery or the repair of major bone breaks. Consequently,in keeping with the autologous nature of the wound or tissue healantcomposition of the present invention, autologous bone from a patient canbe ground or made into powder or the like, and mixed into the plateletconcentrate of the present invention. Serum comprising thrombin is thenmixed in with the platelet concentrate and bone fragments in an amountsufficient to allow the resulting gel to be applied to the desiredlocale where it congeals.

In instances where the desired wound or tissue healant composition ofthe present invention is to further function as a delivery device ofdrugs and proteins with other biologic activities the method of thepresent invention may be modified as follows. Prior to adding the serumcomprising thrombin to the platelet concentrate, a wide variety of drugsor proteins with other biologic activities may be added to the plateletconcentrate. Examples of the agents to be added to the plateletconcentrate prior to the addition of the serum include, but are notlimited to, analgesic compounds, antibacterial compounds, includingbactericidal and bacteriostatic compounds, antibiotics (e.g.,adriamycin, erythromycin, gentimycin, penicillin, tobramycin),antifungal compounds, anti-inflammatories, antiparasitic compounds,antiviral compounds, enzymes, enzyme inhibitors, glycoproteins, growthfactors, recombined (e.g., lymphokines, cytokines), hormones, steroids,glucocorticosteroids, immunomodulators, immunoglobulins, minerals,neuroleptics, proteins, peptides, lipoproteins, tumoricidal compounds,tumorstatic compounds, toxins and vitamins (e.g., Vitamin A, Vitamin E,Vitamin B, Vitamin C, Vitamin D, or derivatives thereof). It is alsoenvisioned that selected fragments, portions, derivatives, or analoguesof some or all of the above may be used.

A number of different medical apparatuses and testing methods exist formeasuring and determining coagulation and coagulation-related activitiesof blood. These apparatuses and methods can be used to assist indetermining the optimal formulation of activator, that is, thrombin,platelet concentrate and plasma necessary to form the wound or tissuehealant compositions of the present invention. Some of the moresuccessful techniques of evaluating blood clotting and coagulation arethe plunger techniques illustrated by U.S. Pat. No. 4,599,219 to Cooperet al., U.S. Pat. No. 4,752,449 to Jackson et al., and U.S. Pat. No.5,174,961 to Smith, all of which are incorporated herein by reference.

The plasma concentrate, PRP, compositions of the present invention canbe admixed with either tricalcium phosphate (TCP), hyaluronic acid (HA),chitosan, cream, cream mask, cell extracts, fat cells, lubricin,cd-gelatin and/or botulinum toxin.

In one embodiment, a plasma concentrate or PRP composition of thepresent invention can be admixed with tricalcium phosphate (TCP),hyaluronic acid (HA), chitosan, cream, cream mask, cell extracts, fatcells, lubricin, cd-gelatin and/or botulinum toxin.

In one embodiment, a plasma concentrate or PRP composition, preferablyof the present invention, can be admixed with tricalcium phosphate(TCP), hyaluronic acid (HA), chitosan, cream, cream mask, cell extracts,fat cells, lubricin, cd-gelatin and/or botulinum toxin.

In one embodiment, a plasma concentrate or PRP composition, preferablyof the present invention, can be admixed with thrombin, preferably athrombin serum of the present invention, and further admixed withtricalcium phosphate (TCP), hyaluronic acid (HA), chitosan, cream, creammask, cell extracts, fat cells, lubricin, cd-gelatin and/or botulinumtoxin.

In one embodiment, a plasma concentrate or PRP composition, preferablyof the present invention, can be admixed with thrombin, preferably athrombin serum of the present invention, and further admixed with a cellextract and tricalcium phosphate (TCP), hyaluronic acid (HA), chitosan,cream, cream mask, other cell extracts, fat cells, lubricin, cd-gelatinand/or botulinum toxin.

Automated apparatuses employing the plunger technique for measuring anddetecting coagulation and coagulation-related activities generallycomprise a plunger sensor cartridge or cartridges and a microprocessorcontrolled apparatus into which the cartridge is inserted. The apparatusacts upon the cartridge and the blood sample placed therein to induceand detect the coagulation-related event. The cartridge includes aplurality of test cells, each of which is defined by a tube-like memberhaving an upper reaction chamber where a plunger assembly is located andwhere the analytical test is carried out, and a reagent chamber whichcontains a reagent or reagents. For an activated clotting time (ACT)test, for example, the reagents include an activation reagent toactivate coagulation of the blood. A plug member seals the bottom of areagent chamber. When the test commences, the contents of the reagentchamber are forced into the reaction chamber to be mixed with the sampleof fluid, usually human blood or its components. An actuator, which is apart of the apparatus, lifts the plunger assembly and lowers it, therebyreciprocating the plunger assembly through the pool of fluid in thereaction chamber. The plunger assembly descends by the force of gravity,resisted by a property of the fluid in the reaction chamber, such as itsviscosity. When the property of the sample changes in a predeterminedmanner as a result of the onset or occurrence of a coagulation-relatedactivity, the descent rate of the plunger assembly therethrough ischanged. Upon a sufficient change in the descent rate, thecoagulation-related activity is detected and indicated by the apparatus.

The wound or tissue healing compositions, haemostatic agents, cellcompositions, isolated cell compositions, cell preparations or cellextracts described herein may be combined with a platelet concentrate(PRP).

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of keratinocytes.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an autologous extract of keratinocytes.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of skeletal muscle cells such as muscle progenitor cellsor satellite stem cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of fibroblasts.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of adipocytes, preadipocytes, pre-endothelial cells, ormesenchymal stem cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of chondrocytes.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of stem cells such as umbilical cord stem cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of tendon cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of periosteal filter or gingival cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of corneal cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of bone marrow cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of osteoblast cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of Schwann cells.

In another further embodiment, the invention provides a process for thepreparation of a wound or tissue healing composition, a haemostaticagent, a cell composition or a cell preparation wherein the cell extractis an extract of pancreas islet cells.

In another further embodiment, the isolated platelet concentratecomposition, the wound or tissue healant composition, the thrombinenriched serum, the cell extract, the wound or tissue healingcomposition, the haemostatic agent, the cell composition and/or the cellpreparation of the invention is/are autologous.

In a further aspect, the present invention provides a kit adapted fortissue regeneration according to the invention wherein the kit furthercomprises separate vials containing calcium gluconate, albumin,chitosan, cream, lubricin. TCP, ETOH and/or CaCl2, syringe holders,clumper and/or a tip applicator with a dual exit. The use of autologousthrombin serum according to the invention has the advantage thatadditives like ETOH and CaCl2 are not required for the preparation of awound healant or PRP preparation.

In another embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell extract provided under step d) or e) is obtained by a processcomprising the steps of:

(A) Providing the said cells in a platelet concentrate according to theinvention,

(B) Optionally culturing the cells, and

(C) Re-suspending the cultured cells obtained under step (B) into aplatelet concentrate according to the invention.

In a further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell expansion under step (A) is performed in a platelet concentrateaccording to the invention such as the final concentration in plateletsis comprised between about 5% and about 40% of the volume of the culturemedium.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) comprises at least one step of plating the cells,for example on a cell culture surface such as a Petri dish or a cultureflask.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention comprisingat least one further step of harvesting the cells after the cell culturestep (B).

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) is performed at 37° C.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) is performed under a gas flow comprising oxygen orair and carbon dioxide. Typically the gas flow comprises 95% of oxygenor air and 5% carbon dioxide.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) lasts for about 3 up to about 4 weeks.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention whereinduring the cell culture step (B), the cell culture medium is regularlychanged during incubation, typically every about 3 days.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) comprises at least one exposure step of the cellsto visible light, typically at about 633 nm, during about 10 minutes. Inanother aspect, the exposure step to visible light is repeated once aweek during cell incubation.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell composition is a keratinocyte or fibroblast cell composition andthe cell culture step (B) comprises at least one exposure step of thecells to visible light, typically at about 633 nm, during about 10minutes. In another aspect, the exposure step to visible light isrepeated once a week during cell incubation.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) comprises at least one step of addition of dilutedplatelet concentrate according to the invention such as the finalconcentration in platelets comprised between about 5% and about 40% ofthe volume of the culture medium.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell composition is a keratinocyte or fibroblast cell composition andthe cell culture step (B) comprises at least one step of addition ofdiluted platelet concentrate according to the invention such as thefinal concentration in platelets comprised between bout 5% and about 40%of the volume of the culture medium.

In another embodiment, the present invention provides an isolated cellcomposition obtainable from a process according to the invention.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a fat cellcomposition such as an adipocyte cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a muscle cellcomposition such as a myoblast cell or a satellite stem cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a corneal cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a cartilage cellcomposition, such as a chondrocyte cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a skin cellcomposition, such as a fibroblast cell or keratinocyte cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a periostealfilter or gengival cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a tendon cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a stem cellcomposition, such as an umbilical cord stem cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a bone marrow cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a Schwann cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a pancreas isletcell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is an osteoblast cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein cells are at a concentration of about 3×10⁵ toabout 10⁶ cells/ml of plasma or enriched plasma.

In another embodiment, the present invention provides compositions,methods and uses for promoting wound or tissue sealing and/or tissueand/or bone regeneration in a wound of a human or a lower animal asdescribed herein.

In another further embodiment, the present invention providescompositions, methods and uses for promoting wound or tissue sealingand/or tissue and/or bone regeneration in a wound of a mammal,preferably human. In another embodiment, the present invention providescompositions, methods and uses for inducing periodontal regeneration ina wound or a periodontal defect of a mammal with periodontal disease orother condition as described herein.

In another further embodiment, the present invention provides a methodfor inducing periodontal regeneration in a wound or a periodontal defector cavity of a mammal with periodontal disease or other conditionwherein the mammal is human.

In another further embodiment, the present invention provides a methodfor inducing periodontal regeneration in a wound or a periodontal defector cavity according to the invention wherein the said therapeuticallyeffective amount of the said wound or tissue healant composition isapplied in a form of semi-solid gel or a growth matrix to the said woundor said periodontal defect or cavity such as described for example inGarg et al., 2000, Dental Implantology Update, 11(6), 41-44.

In another embodiment, the present invention provides a method forpromoting skin tissue regeneration in a scar or wrinkle as describedherein.

In another embodiment, the present invention provides a method forinducing myocardial regeneration according to the invention, wherein thesaid therapeutically effective amount of the said muscle cellcomposition according to the invention is injected in combination with aplatelet concentrate (PRP) into the myocardium, typically into the leftventricule myocardium. Injection can be made as direct injection ormultiple catheter injection. Myoblasts or satellite cells can beengineered ex vivo as described in the present description onto ade-epithelised and UV irradiated human biological amnion andbiocomposite construct, as a monolayer in the present description. Theamnion is then sutured to the ischaemic epicardium in order torepopulate the underlying tissue with stem cells, in order to improvethe contractile power of the ventricular wall and myocytes.

In another embodiment, the present invention provides a method forinducing myocardial regeneration according to the invention, wherein thesaid therapeutically effective amount of the said muscle cellcomposition according to the invention is injected in combination with aplatelet concentrate (PRP) into the myocardium, together with atherapeutically effective amount of fibroblast cell compositionaccording to the invention in combination with a platelet concentrate(PRP). In another embodiment, the present invention provides a methodfor inducing myocardial regeneration according to the invention, whereinthe said therapeutically effective amount of the said muscle cellcomposition according to the invention is applied in combination with aplatelet concentrate (PRP) on the ventricular surface in the form of anamnion patch preferably incubated into a myoblast and satellite stemcell composition according to the invention.

In another embodiment, the present invention provides a method forinducing corneal regeneration according to the invention, wherein thesaid therapeutically effective amount of the said corneal cellcomposition according to the invention in combination with a plateletconcentrate (PRP) is applied to the corneal tissue in the form of anamnion patch preferably spread on a dissolvable contact lens.

Said method of treating a wound, a tissue or a disease may include theuse of any of the compositions described herein; it may also include theuse of any composition made by any of the methods described herein.

The methods, the devices and the kit according to the invention presentthe advantages to provide a time-effective and relatively low-cost wayof obtaining platelet concentrates in a single operation that is easy toimplement and adapted to a point-of-care application. The methods of theinvention present the advantage to not only lead to enrichedpreparations wherein the platelets are concentrated in such a high yieldbut also wherein the content in erythrocytes is low. The compositions ofthe invention present the advantage of having a high content inplatelets, a low content in erythrocytes with completely maintainedproperties for its subsequent therapeutic use in-vivo. Morespecifically, the ability of the platelets to release the principalgrowth factors involved in tissue regeneration (PDGF, TGF-beta, IGF,VEGF and EGF) at levels for several days (or the 30 days life span ofthrombocytes) is maintained. In addition, to the extent the compositionsof the invention are made from autologous blood, the invention describedherein reduces the disease transmission and immunoreaction risksassociated with the use of the treatment materials made from biologicalmaterials obtained from one or more third parties. The inventiontherefore provides an improved biological wound healing and tissueregenerating material, preferably autologous, promoting tissue such asskin, cartilage and bone regeneration, especially cicatrisation and/orrejuvenation. The benefits of the invention comprise a simple and rapidmethod of preparation of improved wound healing and tissue regeneratingmaterials adapted to point-of-care services and which proved to decreasethe healing time, associated pain and medical costs. Further, the woundhealing and tissue regenerating material decreases the graft rejectionrisks and improves graft success rates. Further, the improved woundhealing and tissue regenerating materials lead to scars having a muchbetter aesthetic final aspect and to the durable filling of scars andwrinkles.

Typically, cell extracts are obtained from a tissue biopsy wherein thebiopsy is preferably performed the same day the mixture with theplatelet concentrate is done. The size of the biopsies is adapted to theaimed therapeutic purpose and the types of cells used in the preparationof the cell composition according to the invention. Examples of biopsiesare given in the Examples below for different types of tissues. Inanother aspect, the invention provides a tube comprising at least onefilter separating the tube in two parts.

In another aspect, the invention provides a tube separated in two partsby at least and preferably one filter.

In one embodiment the tube is used for collecting and/or separating afluid sample.

Preferably, the two parts differ in size and/or diameter.

In another aspect, the invention provides a tube for collecting andseparating a fluid sample comprising:

i) two distinct parts differing in size and diameter,

ii) at least one filter separating the two parts, and

iii) optionally an anticoagulant.

In a preferred embodiment of the invention, the tube consists of thefeatures as illustrated in FIGS. 1 to 14.

In a preferred embodiment, the tube is made of Cyclic Olefin Polymer(COP) or Cyclic Olefin Copolymer (COC).

In one embodiment, the tube contains an anticoagulant, preferablybuffered sodium citrate solution or an anhydrous sodium citrate.

In a preferred embodiment, the tube contains a buffered sodium citratesolution at 0.10 M or an anhydrous sodium citrate at 3.5 mg/mL.

In a preferred embodiment, the filter is fixed to the tube. Preferably,the filter is prolonged vertically in order to have an extended surfacein contact with the tube (extended on the vertical axis of the tube).This has the advantage of increasing the stability of the filter,further preventing any movement of the filter. In a preferredembodiment, the tube comprises a filter made of two layers. Preferably,the 2 layers are superposed (one above the other; FIG. 4, FIGS. 10 to14). Preferably, the outer layer is prolonged vertically in order tohave an extended surface in contact with the tube (extended on thevertical axis of the tube FIGS. 2, 3, and 5A). Preferably, the innerlayer is also prolonged in order to align with the outer layer. In oneembodiment, the inner layer is not prolonged (only on the horizontalaxis).

In one embodiment, 2, 3, 4, 5 or more filters are comprised in the tube.

In a preferred embodiment, the first part of the tube located in theabove or top portion of the tube is of greater volume than the secondpart located in the below or bottom portion of the tube. In a furtherpreferred embodiment, the first part of the tube comprising the filterhas a volume of about 9.5 cm3 and the second part of the tube has avolume of about 3.5 cm3 (FIG. 5A).

In a preferred embodiment, the volume of the first part of the tubelocated in the above portion of the tube comprising the filterrepresents about 70% of the total volume of the tube. In one embodiment,the volume of the first part of the tube located in the above portion ofthe tube comprising the filter represents about 51%, 55%, 609%, 65%,68%, 70%, 73%, 75%, 80%, 90%, 95% or more of the total volume of thetube. In a further preferred embodiment, the volume of the second partof the tube located in the below portion of the tube represents about30% of the total volume of the tube. In one embodiment, the volume ofthe second part of the tube located in the below portion of the tuberepresents about 49%, 45%, 40%, 35%, 32%, 30%, 27%, 25%, 20%, 15%, 10%or 5% or less of the total volume of the tube.

In one preferred embodiment, the tube has a total volume of about 13 cm3(FIG. 5A).

In a preferred embodiment, the second part of the tube located in thebelow portion of the tube has a smaller diameter than the first part ofthe tube located in the above portion of the tube. This has theadvantage that the filter will stay in its position under centrifugationor any other mechanical stress.

In a preferred embodiment, the external diameter of the first part ofthe tube located in the above portion of the tube is of about 15.5 mm orabout 15.4 mm (FIG. 3). In a preferred embodiment, the internal diameterof the first part of the tube located in the above portion of the tubeis of about 13.32 mm or 13.31 mm (FIG. 3).

In a preferred embodiment, the external diameter of the second part ofthe tube located in the below portion of the tube is of about 13.7 mm(FIG. 5A). In a preferred embodiment, the internal diameter of thesecond part of the tube located in the below portion of the tube is ofabout 11.6 mm.

In a preferred embodiment, a filter made of two layers separates the twoparts of the tube. Preferably, the 2 layers are superposed (one abovethe other: FIG. 4, FIGS. 10 to 14).

A preferred filter is shown in FIG. 4 and FIGS. 7 to 14. Preferably, thefilter comprises an internal and external layer (FIGS. 10, 11 and 13).

In a preferred embodiment, the filter is fixed to the tube. In anotherembodiment, the filter is not fixed to the tube.

In one embodiment, the filter comprises symmetrical series of rangeswith openings disposed as shown in FIGS. 7A, 7B, 8 and 9. Preferably,the filter consists of 4 symmetrical series of ranges with 2 ranges perseries, each range consisting of an opening. For the first range ofopenings located towards the center of the tube, the distance betweenthe internal part of one opening and the internal part of anotheropening is about 5.13 mm (FIG. 4, item 4). For the first range ofopenings located towards the center of the tube, the distance betweenthe external part of one opening and the external part of anotheropening is about 5.67 mm (FIG. 4, point 3). For the second range ofopenings located towards the border of the tube, the distance betweenthe internal part of one opening and the internal part of anotheropening is about 7.73 mm (FIG. 4, item 2). For the second range ofopenings located towards the border of the tube, the distance betweenthe external part of one opening and the external part of anotheropening is about 8.27 mm (FIG. 4, item 1). In one embodiment, thedistance of each opening is about 0.53 or 0.54 mm (FIG. 9 and FIG. 14,item 21).

Preferably, the upper or internal layer consists of 4 symmetrical seriesof ranges with 2 ranges per series (FIGS. 9, 13 and 14), each rangeconsisting of an appropriate number of funnels disposed regularly (FIGS.13 and 14). Preferably, the gap or opening located at the bottom of eachfunnel is of a length of about 0.27 mm (FIG. 14, item 26). Preferably,the distance from one extremity to the other of the funnel is about 1mm, corresponding to the thickness of the upper layer (FIG. 14, item24). Preferably, the thickness of the upper layer is about 1 mm (FIG.12, item 34 and FIG. 14, item 24). The number of funnels will depend onthe size of the layer. Preferably, the funnel comprises a closedaperture at its base (item 26 of FIG. 14).

Preferably, the lower or external layer consists of 4 symmetrical seriesof ranges with 3 ranges per series (FIGS. 8, 13 and 14), each rangeconsisting of an appropriate number of trapezoids disposed regularly(FIGS. 13 and 14). The number of trapezoids will depend on the size ofthe layer. Preferably, each trapezoid is separated by a distance ofabout 0.53 mm (FIG. 14). The base of each trapezoid is of a length ofabout 0.77 mm (FIG. 14). Preferably, the height of each trapezoid isabout 0.5 mm (FIG. 14). Preferably, the thickness of the lower layer isabout 1 mm (FIGS. 12 and 14).

Preferably, the ranges of the upper and lower layers always alternate.Preferably, a first range of trapezoids of the lower layer will befollowed by a first range of funnels of the upper layer which will befollowed by a second range of trapezoids of the lower layer followed bya second range of funnels of the upper layer which will be finallyfollowed by a third range of trapezoids of the lower layer (FIGS. 4, 13and 14).

Preferably, each funnel of the upper layer is located in such a mannerthat the prolongation of the center of the funnel will fallapproximately between two trapezoids of the lower layer (FIGS. 4, 13 and14). More preferably, each funnel of the upper layer is located in sucha manner that the prolongation of the center of the funnel will fallexactly between two trapezoids of the lower layer (FIGS. 4, 13 and 14).Preferably, a trapezoid alternates continuously with a funnel.

When the tube of the present invention is centrifuged, the closedaperture at the base of the funnel (item 26 of FIG. 14) opens due to thecentrifugal force allowing the passage of the Red Blood Cells that aredirected between two trapezoids and finally to the lower section of thetube. Once centrifugation stops, the aperture of the funnels will closehindering the reflux of the Red Blood Cells to the upper portion of thetube. The trapezoids of the lower layer located beneath each funnel willfacilitate the passage of the Red Blood Cells to the lower portion ofthe tube and help in preventing the reflux of the Red Blood Cells.

Preferably, the volume of the upper layer is about 0.26 cm3. Preferably,the volume of the lower layer is about 0.29 cm3.

Preferably, the upper layer is made of polypropylene (PP) (FIG. 10, item6). Preferably, the lower layer is made of a thermoplastic elastomer(TPE) (FIG. 10, item 7). Other components may be used as described inthe present invention or known by the skilled artisan.

Preferably, the center of the filter is curved (FIGS. 4, 10 to 13). Thishas the advantage of canalizing the whole blood towards the funnels.Preferably, the filter is curved in such a manner as to have a length ofabout 0.5 mm from the center of the tube to the base of the lower layer(FIG. 12, item 36). Items 30 and 32 in FIG. 12 are injection points.

Preferably, the height of the lower layer is about 8 mm (FIG. 2 and FIG.10, item 14). Preferably, the outer diameter of the lower layer is about12.74 mm (FIG. 10, item 17). Preferably, the inner diameter of the upperlayer is about 9.6 mm (FIG. 10, item 16). Preferably, the height of thelayer from the upper non-curved surface of the upper layer is about 6 mm(FIG. 10, item 9). Preferably, the height of the layer from the centerof the curved upper surface of the upper layer is about 5.5 mm (FIG. 10,item 11).

In one embodiment of the invention, the tube comprises only a filterwith funnels.

In another embodiment of the invention, the number of funnels and/ortrapezoids can vary. In another embodiment of the invention, the numberof ranges of funnels and/or trapezoids can vary. In another embodimentof the invention, the number of symmetrical series of ranges of funnelsand/or trapezoids can vary. In another embodiment of the invention, thetube consists of 2 symmetrical series of ranges of funnels and/ortrapezoids.

The present invention also encompasses a tube wherein the lengths of allcomponents differ from those indicated herein. The present inventionalso encompasses a tube wherein the lengths of all components differfrom those indicated herein with a length of about 0.1 mm, 0.2 mm, 0.3mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 5 mm, 8 mm, 10 mm, 15 mm, 20 mm, 50 mm,75 mm, 1 cm, 2 cm, 5 cm, 10 cm or more.

In one embodiment, the filter is shaped or modeled with a laser (FIG.14, item 37).

The tubes of the present invention have the advantage of separating withhigh efficacy Red Blood Cells from the plasma. Using the tubes of thepresent invention, the percentage of RBCs in the plasma is reduced bymore than 99%, 98%, 95%, 93%, 90%, 85%, 800/%, 75%, 706, 65% or 60%compared with the percentage of RBCs present in whole blood.

COCs or COPs, apart from cost savings and being a stiff and strongmedical material, have the advantage that its transparency is similar toglass in its natural form. Typical COC material will have a highermodulus than HDPE (High-density polyethylene (HDPE) or polyethylenehigh-density (PEHD) is a polyethylene thermoplastic made from petroleum)and PP (Polypropylene or polypropene (PP) is a thermoplastic polymer).COC also has a high moisture barrier for a clear polymer along with alow absorption rate. In medical applications, COC is noted to be a highpurity product with low extractables. COC is also a halogen-freeproduct.

COC material has excellent optical clarity and a high barrier to watervapor. It molds fine features with great fidelity, withstands all commonsterilization methods, and resists hydrolysis and a wide range ofchemicals.

COC material also has good heat resistance, mechanical properties,hardness, dimensional stability, and electrical insulating properties.Because it is very low in extractables and has excellent in-vivo andin-vitro biocompatibility, it meets USP Class VI and ISO 10993requirements and has received FDA Drug and Device Master File numbers.

In addition, COC, with its transparency, stiffness, low weight, heat andchemical resistance, biocompatibility, dimensional stability to moisturebarrier (low moisture permeability), moldability, and lack of halogens,offers many benefits in use. It reduces breakage in devices andpackaging compared to glass, extends medication shelf life, allowsdiagnostic readings at UV wavelengths, and enables smaller and fasterdiagnostic equipment.

In addition, COC grades can also undergo sterilization by gammaradiation, steam and ethylene oxide and can thus be sterilized by allcommon methods.

In another aspect, the invention provides a blood bag system or bloodcollection tubes device according to FIG. 15.

Blood collected and/or stored in a blood bag system or blood collectiontubes device according to the invention may be used in any of theprocesses, compositions, products, and uses according to the presentinvention.

The blood collection tubes or bags may be evacuated and/or sealed. Theblood collection tubes or bags may be filled with thixotropic gel and/oranticoagulant.

The blood bag system or blood collection tubes device of the inventionconsists in a multi-channel blood collector allowing delivery of plasmaand cell components to multiple bags or tubes.

In another aspect, the invention provides a blood bag system or bloodcollection tubes device comprising a single entry conduit connected to amultiple conduit adapter with adapter conduits connected to at least twobags or tubes, wherein each adapter conduit of the multiple conduitadapter is connected to one single bag or tube.

In another aspect, the invention provides a blood bag system or bloodcollection tubes device comprising:

a) a single entry conduit,

b) a multiple conduit adapter with adapter conduits, and

c) at least two bags or tubes,

wherein the single entry conduit is connected to the multiple conduitadapter and wherein each adapter conduit of the multiple conduit adapteris connected to one single bag or tube.

In one embodiment, the invention provides a blood bag system or bloodcollection tubes device according to the invention for pooling bloodcomponents.

Preferably, the blood bag system or blood collection tubes devicecomprises an entry conduit (1), a multiple conduit adapter (2), adapterconduits (3) and at least two bags or tubes (4) as illustrated in FIG.15.

In one embodiment, the blood bag system or blood collection tubes devicefurther comprises one or more fixation units (5) (FIG. 15). Preferably,one fixation unit is provided for each adapter conduit. In oneembodiment, two or more fixation units are provided for each adapterconduit.

Preferably, each bag of the blood bag system or blood collection tubesdevice further comprises a cap. FIG. 15 illustrates a blood bag systemor blood collection tubes device wherein each bag (4) comprises a cap(6). Preferably, the cap can be unlocked or unscrewed in order toconnect a sterile syringe. The cap has the advantage of allowing easyaspiration of the content of the bag in sterile conditions. In oneembodiment, a Luer-Lock syringe may be used. Alternatively, a needle maybe used instead of a cap.

Preferably, the entry conduit and adapter conduits are flexible.Preferably, the entry conduit and adapter conduits are tubular.

In one embodiment, the blood bag system or blood collection tubes devicecomprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more bags ortubes. Preferably, the blood bag system or blood collection tubes devicecomprises 8 bags or tubes.

Preferably, the invention provides disposable bags or tubes for singleuse.

In one embodiment, each bag may contain up to and about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 25, 30 ml or more of blood.Preferably, each bag contains about 10 ml of blood. Preferably, thevolume of each bag is adapted for single use. Preferably, the amount ofblood collected in each bag is in a sufficient quantity for one singleuse.

Preferably, the blood bag system or blood collection tubes deviceprovides a sealed flow communication. More preferably, the blood bagsystem or blood collection tubes device provides a sealed and sterileflow communication.

Preferably, each bag can be individually sealed once the bags or tubesare filled. This has the advantage of having individual, ready-to-use,single disposable bags or tubes.

Preferably, each bag is submitted to a vacuum step. Preferably, theblood bag system or blood collection tubes device is submitted to avacuum step.

In one preferred embodiment, each bag is inserted in a second protectionor envelope. This has the advantage of offering a double protection inconformity with ISO standards on packaging (ISO 11607-1 and/or ISO11607-2).

Preferably, each bag is inserted in the second envelope after vacuum.Preferably, the second envelope is submitted to vacuum and sealed.Sterilization, vacuum steps and double protection offer bags or tubesthat are very safe to use.

In one embodiment, fluid flow may be controlled by conventional valvingmeans such as snap-off plugs, removable plugs, or slide clamps.

The blood bags or tubes system may be of conventional construction beingmade of a plastic material that is blood compatible, flexible,translucent, and sterilizable. The plastic may be a polyvinylchloride,polyester, polyolefin, polyurethane, and so forth and may include blendsof the above materials. The entry conduit and/or adapter conduits may bemade of a plastic material that is the same as or different from theplastic material of the blood bags or tubes.

In one embodiment, filtering means may be integrated to the blood bagsystem or blood collection tubes device of the invention. The filteringmeans may include a housing made of rigid polyvinylchloride or the likeand tubing fitments. Filtering means may be filled with a filtrationmedium such as cotton wool or cellulose acetate or other syntheticfibers such as polyester, polyamides, and the like. The amount offiltration medium depends upon the amount of red cells to be filtered.Usually about 20-50 grams of filtration medium are employed per 200-250ml of red cell concentrate.

In one embodiment, an additive solution is added to prolong the storagelife of the red cells. This additive solution may be, for example, aconventional red cell storage solution such as that described inGinzburg et al., Bibl. Haemotol., 1971, No. 3, Pt. 2, 217-220; Wood etal., Blood, Vol. 42, No. 1, 1973, 17-25; Beutler, “The Red Cell inVitro”. Grum and Stratton, New York, N.Y., 1974, p. 201; Lovric et al.,Medical Journal of Australia. Vol. 2, 183-186, 1977: U.S. Pat. No.4,267,269; in an amount of about 50-100 ml per 200-250 ml of red cellconcentrate.

In one embodiment, the bags or tubes of the blood bag system or bloodcollection tubes device of the invention may contain an anticoagulantsuch as Adenine-Citrate-Dextrose (ACD), Citrate-Phosphate-Dextrose(CPD), Citrate Phosphate-277 millimoles Dextrose (CP2D), CPD plusadenine, or other conventional anticoagulant, with which the collectedblood mixes. The collected blood may then be processed directly orstored usually at about 4°-6° C. At processing, the bag system may becentrifuged as is customary in the art causing the red cells in theblood to settle at the bottom of the bag. Blood plasma is expressed byconventional techniques which fresh plasma and a platelet concentrate.

The blood contained in the bags or tubes may be used in any aspectsand/or embodiments of the present invention.

The blood bag system or blood collection tubes device is easilymanufactured and produced with great utility in the field of medicine.By using this invention, blood collections are easily made, become saferand more efficient. Advantageously, the invention provides easiertraceability. Advantageously, the invention provides a blood bag systemor blood collection tubes device and/or kit and/or device comprising theblood bag system or blood collection tubes device that allows easy,rapid and safe collection, storage and delivery of any blood component.A further advantage is that the collection, storage and delivery of theblood component are performed under sterile conditions in conformitywith ISO standards. A further advantage is that the blood bag system orblood collection tubes device and/or kit and/or device are particularlyadapted for a use at the point of care.

Preferably, the collection, storage and delivery of the blood componentis performed under sterile conditions.

In one embodiment, a valve is provided which may be disposable, using amaterial such as acrylic resin in its manufacture.

In another aspect, the invention provides a sterile kit and/or devicecomprising at least one blood bag system or blood collection tubesdevice according to the invention. All of the apparatus required to poolblood components is provided in a sterile kit, making the pooling ofblood components safe and economical.

The blood provided by the blood bag system or blood collection tubesdevice and/or kit and/or device according to the invention may be usedfor the preparation of thrombin, platelet rich plasma, plateletconcentrate, cell extract, cell composition, wound healing agent and/ortissue healing agent or haemostatic agent or biological glue whetheraccording to any aspects and/or embodiments described herein.

The blood provided by the blood bag system or blood collection tubesdevice and/or kit and/or device according to the invention may be usedfor all the applications described herein. For example, the bloodprovided by the blood bag system or blood collection tubes device and/orkit according to the invention may be used in surgery, ambulatoryconditions or on chronic wounds.

The blood provided by the blood bag system or blood collection tubesdevice and/or kit and/or device according to the invention may be usedas a homologous or autologous component.

When used as a homologous component, the blood and/or blood componentsprovided by the blood bag system or blood collection tubes device and/orkit and/or device according to the invention represents an advantageousefficient, simple and low-cost alternative to known systems. Itrepresents when the collection of autologous blood is not possible.

Examples illustrating the invention will be described hereinafter in amore detailed manner and by reference to the embodiments represented inthe Figures.

EXAMPLES

The following abbreviations refer respectively to the definitions below:

ATS (autologous thrombin serum); BU (Baxothrobin unit); DMEM (Dulbecco'sminimum essential medium); DMSO (Dimethyl Sulfoxide); EC (Enrichedclot); FCS (fetal calf serum); HT (healing time); IU (InternationalUnit); PBS (Phosphate Buffered Saline); PET (polyethyleneterephthalate); PRP (platelet-rich plasma); PPP (platelet-poor plasma);USP (United States Pharmacopoeia); cm (centimeter); dL (decilitre); g(gram); Gy (gray); J (Joule); L (liter); min (minute); mm (millimetre);M (molar); mL (millilitre); nm (nanometre); rpm (Rotation per minute);Vol. (volume).

General Procedures & Conditions

To determine the effectiveness of compositions of the invention inpromoting wound healing and/or bone and/or tissue regeneration, thefollowing experiments are performed. Whole human blood samples arecollected in a separator tube according to the invention. A tube thatmay be used is for example an approximately 10 to 15 mL glass tube (16mm diameter and 120 to 130 mm in length) containing 2 to 3 mL ofpolyester-based thixotropic gel as well as 1 mL of sodium citratesolution at 0.1 M and containing a usable vacuum of or about 8.0 mL to10 mL. This tube constitutes a ready-to-use device for the preparationof a platelet concentrate composition of the invention.

Another example of tube that may be used is a tube of approximately 10mL in PET (polyethylene terephthalate) containing 2 mL of a thixotropicgel comprised of a polymer mixture and a sodium citrate solution (about0.1 M) and containing a usable vacuum of about 8 mL to 10 mL,constitutes a ready-to-use device for the preparation of a plateletconcentrate according to the invention. Another example of tube that maybe used is a tube according to the invention. These tubes are filledwith 2 components, vacuum aspirated, sterilized by irradiation (such asprescribed by ISO 11137, UNI EN ISO 11737-2, UNI EN 552, UNI EN 556) andhermetically sealed by a traditional cap such as a mottled bromobutylconventional rubber stopper for the glass tube and a chlorobutylestopper having a polyethylene cover for the operator safety. Then, thetube is centrifuged at or about 1500 g up to or about 2000 g for about 3to 10 min, i.e., of or about 2,500 rpm up to or about 3,000 rpm with acentrifuge with a swinging rotor, having a radius of 14 cm. In case of acentrifuge having a rotor with a fixed angle of about 45°, thecentrifugation time should last from 5 to about 15 min. Alternatively,the tube is centrifuged according to the invention.

After centrifugation, the platelet concentrate is collected for use intherapeutic or cosmetic applications of the invention or for thepreparation of further compositions containing the obtained plateletconcentrate through the mixture with further agents such as cellextracts, preferably autologous (e.g., keratinocytes, fibroblasts, bonemarrow cells, osteoblasts, chondrocytes, myoblasts, corneal cells,Schwann cells, fat cells, umbilical cord stem cells, tendon cells,pancreas islet cells, ligament and gingival cells, periosteal membranecells) and/or bone substitutes and/or coagulation activators.

The plasma concentrate, PRP, compositions of the present invention canbe admixed with either tricalcium phosphate (TCP), hyaluronic acid (HA),chitosan, cream, cream mask, cell extracts, fat cells, lubricin,cd-gelatin and/or botulinum toxin.

For the preparation of cell compositions, according to the invention,the cells are prepared according to the general protocol as follows:

a) Biopsy

A biopsy of the corresponding tissue is obtained under sterileconditions using standards methods adapted to the specific cell thatwill be collected. The cells can be purified by washing, centrifugationor sedimentation. The cells can be isolated by centrifugation, byenzymatic digestion (trypsin, collagenase or recombined trypsin). Thecells are used extemporaneously or optionally after ex-vivo culture andcell proliferation as follow.

b) Ex-Vivo Culture and Cell Proliferation

Cells used for the preparation of cell compositions, such askeratinocytes, bone marrow cells, fibroblasts; periosteum or cornealcells, such as corneal limbal stem cells, melanocytes and Langerhanscells; fat cells; muscle cells such as myoblasts and satellite cells;osteoblasts; chondrocytes; umbilical cord cells; Schwann cells,mesenchymal stem cells (MSCs), preadipocytes, pre-endothelial cells,tendon or pancreatic cells are traditionally expanded in a cell carriermedium (e.g., DMEM or Ham's) on plates (e.g., Petri dishes or cultureflask) coated with a platelet concentrate, preferably autologous,enriched with fibronectin. Advantageously, a cell carrier medium likeDMEM or Ham's is not required with the use of a plasma concentrate orPRP of the present invention, at a concentration of about 5% to about20%. In addition, advantageously, this makes the cell preparations 20%more effective. The cell preparations may eventually be enriched withfibronectin. The culture media may be enriched preferably with DMEM forexample in the case of keratinocytes. For cells such as boneosteoblasts, chondrocytes and myoblasts, enzymatic digestion of thecorresponding tissue in presence of for example collagenase or trypsinis necessary before plating. Incubation on the plates is performed at37° C. under a gas flow of 95% oxygen or air and 5% carbon dioxide.

Typically, incubation times vary from 10 to 20 min. The cell expansionmay eventually be enhanced (like for example in the case of myoblast,fibroblast and chondrocyte cells) by phototherapy (e.g., light exposureat 633 nm of about 10 min at 2 J/cm², once a week during the incubationphase).

The explants may be cultured in Petri dishes or culture flask usingair-lifting technique (Molnar et al., 1996, Tissue & Cell, 28:547-556)and air interface method (Meller et al., 2002, Br. J. Opht., 86,463-471) with half of the explant exposed to air. The culture medium ischanged regularly during incubation, such as every 3 days. The expansionof the cells in a 2D mode as planar monolayers, is obtained for examplefor myoblast, fibroblast and chondrocyte cells. A 3D cell growingpattern can be obtained for example for corneal, myoblast, fibroblast,chondrocyte, adipocyte and keratinocyte cells, by adding dilutedautologous platelet concentrate composition according to the inventionat about 5 to about 40% volume of plasma or enriched plasma to theculture medium. Typically, the addition of diluted autologous plateletconcentrate composition according to the invention may be performed twoor three times during the incubation time. The 3D biological scaffoldthen obtained allows to enhance the extra-cellular matrix which isuseful for autologous stem cell transfer.

After incubation, cells may be released from dishes with gentle trypsindigestion that lifts off the cells and allows them to be pelleted.Alternatively, the cells are made thermosensible and cells may bereleased by heat (may use a Petri Dish coated by a thermosensitivepolymer).

c) Cell Quality and Safety Check

The cell viability in the so-obtained cell preparation is checked bymicroscopic cell count, flow-cytometer cell count together withimmunochemistry on tissue markers by standard techniques. Cell viabilityis also tested via trypan-blue just after cell release by trypsin.Safety of the preparation is also checked through contamination checkvia microbiology assay to exclude contamination with viruses or bacteriaand to avoid transfer of zoonotic infections. The use of Fetal CalfSerum (FCS) is avoided thus preventing transmission of Mad Cow Disease.

d) Administration of the Cell Preparation

The cell preparation obtained above is placed in autologous plateletconcentrate composition according to the invention eventually as cellcarrier vehicle for transport before delivery to the patient. Then, thecell preparation obtained above is injected or transplanted into thepatient. The injection or transplantation mode has to be adapted to thetype of cells contained in the cell preparation according to theinvention and to the aimed therapeutic or aesthetic effect. More detailsare given in the Examples below on the method for the preparation anduse of the cell compositions according to the invention morespecifically, depending on the type of cells and aimed therapeutic oraesthetic effect.

Keratinocyte cell or fibroblast cell preparations according to theinvention may be used readily after collection or after cell culture asdescribed above. However, the cell preparations according to theinvention may be prepared after cell culture as described above.

The cell preparations according to the invention present a betterviability and stability (including integrity of cell propertiespreserved such as ability to synthesize proteins and deliver growthfactors) than cells prepared in a medium without autologous plateletconcentrate composition according to the invention. Further, cellproliferation so obtained is enhanced: cells grow faster (about 2 to 5days quicker) and are denser compared to control mediums and serumstarved media. The advantage of the process for the preparation of acell composition according to the invention is that the same autologousmedium is used as vector for cell culture, cell preservation, cellinjection, vector for cell bio-stimulation and tissue regeneration.

Example 1

Therapeutic use of the autologous platelet concentrate of the inventionin combination with an autologous thrombin enriched serum An autologousthrombin serum to be used as a thrombin enriched preparation is preparedaccording to the invention.

One of the original features of this process is that the separator tubesof the invention containing respectively the autologous thrombin serumpreparation and the platelet concentrate preparation can be preparedsimultaneously. The glass tube for the preparation of autologousthrombin serum (ATS) is centrifuged about 8 minutes to about 10 minutes.The plastic tube (COC or COP) or glass tube for the preparation of aplasma concentrate or PRP composition is centrifuged about 8 minutes toabout 10 minutes. Hence, advantageously the ATS and PRP composition areready for use at approximately the same time.

To allow the polymerization of fibrinogen into a fibrin mesh (whichoccurs during the coagulation process) to occur only at the moment ofapplication of the platelet-rich preparation on the wound, the plateletconcentrate composition and autologous thrombin serum (coagulationactivator) are applied simultaneously at a vol. ratio of about 10:1,10:3 to about 10:10 (concentrate to coagulation activator ratio) to thewound, depending on the application. The difference in proportion altersthe rapidity of coagulation for the glue effect.

The simultaneous delivery of both preparations is achieved for exampleby a device comprising two syringes (e.g., 10-mL syringe for theplatelet concentrate composition and a 1-mL or 3 mL syringe for thethrombin serum), that releases the preparations simultaneously so thatthey mix and polymerize upon contact with the wound.

Example 2

Cosmetic use of the autologous platelet concentrate of the inventionExamples of cosmetic use of the autologous platelet concentrate of thepresent invention include:

Admixing the platelet concentrate according to the invention with acream, preferably an emulsion, before application to a wound, aftersurgery or on healthy skin. During the absorption process, the plateletpreparation is carried into the skin by the cream or emulsion in orderto amplify the hydrating benefit and to bio-stimulate the regenerationor rejuvenation of the skin. Injection in the dermal layer may be madewith the technique of mesotherapy by repeated injections by hand or adevice without needle. Injection may be made subcutaneously in thewrinkle for volume correction. Injection of the platelet concentratecombined at a ratio of 10:1 or 10:3 with the autologous thrombin serumfor filler effect and/or consistent volume correction may be made in thewrinkle, forehead, jowls, molar region, cheeks, chin, neck and/or chest.When appropriate, injection of plasma concentrate combined to a freshlyaspirated fat tissue at a ratio of about 10:10 may be madesubcutaneously for important volume correction of the face. Whenappropriate, injection of plasma concentrate combined to a freshlyaspirated fat tissue at a ratio of about 10:3 or about 10:2 may be madesubcutaneously for important volume correction of the breast orcontouring.

Using a hydrogel like the Albugel (EP 1 543 846) preparation of 100%Albumin or any other hydrogel resulting from the reticulation of Albuminand other chemical compound like polyethylene glycol or any otheringredient, using a paper based highly hydrophilic carrier to leave incontact with the skin until the platelet rich plasma is absorbed.

The present invention encompasses the following mesotherapy injectionmethods:

-   -   In the papillary dermis,    -   Subcutaneous in the reticular dermis, or    -   In a deep level above the periosteum.

The 3 levels of injection also called “Medical face lifting” may beperformed with plasma alone, plasma plus adipose tissue and/or plasmaplus tricalcium phosphate.

Example 3: Autologous Muscle Cell Association Preparation

Examples of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinskeletal muscle cells (muscle progenitor cells or satellite stem cells)are provided under step (d) or (e).

a) Myoblast Progenitor Stem Cells

Skeletal muscle biopsy is obtained from the Vastus lateralis andmeasures 7×3 cm. Muscle is primed the day before biopsy, withintra-muscular injection at proposed biopsy site (10 by 15 cm skin areaon lateral aspect of thigh overlying the vastus lateralis muscle andjust above the knee joint, on either side) with Decadon and Marcaine(long acting Lignocaine). Muscle is diced and enzymatically digestedwith combination of collagenase, pronase and trypsin (Worthington).Muscle explants are plated in Petri dishes coated with an autologousplatelet concentrate composition according to the invention, andincubated in 95% oxygen and 5% carbon dioxide at 37° C. for 3 to 4weeks. Desmin or CD-56 expression is used as myoblast marker to identifymyoblasts from fibroblasts. Myoblast progenitor cell proliferation in 3Dis shown on FIG. 3. Cell proliferation can be enhanced by photo-lightexposure at 633 nm of 2 J/square centimetre for 10 min during culture.The day of transplantation (e.g., after 3 to 4 weeks of incubation), theskeletal muscle cells may be released by trypsin or any method asdescribed herein and placed in the autologous platelet concentratecomposition according to the invention. Injections into the myocardiumcan be made as direct injection or multiple catheter injections into theleft ventricle myocardium. The myoblast cell preparation according tothe invention is useful for cardiac disorders such as heartregeneration, treatment of heart failure, chronic cardiac failure,ischemic and non-ischemic cardiac failure and non-ischemiccardiomyopathy. Ejection fraction can be improved by 9% for cardiacrecipients of skeletal myoblasts.

The above cell preparation may also be useful for the treatment ofurinary incontinence (myoblast cell extracts prepared as described aboveand injected into the bladder neck), reflux oesophagitis orgastro-oesophageal reflux disorder (myoblast cell extracts prepared asdescribed above injected into the lower oesophageal sphincter) and analincontinence (myoblast cell extracts prepared as described above andinjected in para-anal area).

Alternatively, a combined preparation of fibroblast and myoblast may becarried out (fibroblasts are present in the muscle biopsy and sproutfrom the perimysium alongside the myotubes and satellite stem cells). Incase of the treatment of cardiac disorders, a mix of fibroblast cellpreparation and myoblast cell preparation (obtained as indicated above)is inserted into the myocardium in a ratio fibroblast-myoblast of about30:70.

For bladder neck incontinence treatment, a separate culture offibroblasts is made at the same time as the myoblasts as described aboveand the fibroblast cell preparation is injected para-urethrally andmyoblast cell preparation is injected into the rhabdosphincter, underultrasound control.

b) Satellite Stem Cells

Myoblasts and satellite stem cells are cultured ex vivo in presence ofautologous platelet concentrate composition according to the invention.Cell proliferation priming is observed after 7 days of primary culture.

Cells are then harvested after incubation of about 3-4 weeks and placedin tissue culture medium (DMEM plus 5-20% vol. autologous plateletconcentrate composition according to the invention) containing a humande-epitheliased amnion patch measuring 4×4 cm and the autologousplatelet concentrate composition according to the invention.

Alternatively, a patch of autologous activated fibrin polymer may beused. The autologous activated fibrin polymer is prepared bycentrifugating the plasma with calcium gluconate at a ratio of about10:3 or about 10:10. The preparation is then subjected to UV irradiationfor 10 min. During incubation (typically about 2 to about 3 weeks), thecells spread over the amnion construct and form a monolayer. Viabilityand monolayer progress is assessed by twice weekly biopsy of patch edgeand histological assessment for thickness of monolayer.

The day of transplantation (e.g., after about 3 to 4 weeks ofincubation), the ventricular surface is spread with the autologousplatelet concentrate composition according to the invention and then thepatch obtained above is placed with cells down side onto a raw surfaceof the ischemic ventricle in order to allow the stem cells on the patchto populate the ischemic segment after ventricular injection. Cellretention is maintained by the amnion that is inert and induces noimmunological reaction.

The satellite stem cell preparation according to the invention is usefulfor heart regeneration and treatment of heart failure as tissueengineering preparation for cardiomyoplasty.

Example 4: Autologous Fibroblast Cell Association Preparation

An example of autologous fibroblast cell association according to theinvention can be prepared by using the process according to theinvention wherein dermal fibroblast cells are provided under step (d) or(e).

Dermal fibroblasts are isolated and expanded according to the followingprocedure: One month before biopsy, the prime donor skin area (behind anear of anterior axillary fold, e.g., non solar aged area) is treatedwith vitamin A cream to activate the dermal fibroblasts. A skin biopsyof 10×6 mm full thickness is performed and dissected under microscope toremove all epithelium. The de-epithelialized biopsy (dermis) is then cutinto 3×3 mm blocks as explants. The papillary dermis is then placedupwards and cultured using air-lifting technique (Molnar et al., 1996,above) and air interface (Metier et al., 2002, above) with half of theexplant exposed to air. The explants are plated (e.g., 6 explants perwell) in DMEM and cultured at 37° C. at 95% oxygen and 5% CO2 for about3-5 up to about 9 days in Petri dishes or culture flask. The medium ischanged every 3 days. The fibroblasts expansion in 2D mode as planarmonolayers, as static growth is observed during incubation. At days 7 to9 after the start of incubation, a change in proliferation and phenotypepattern to 3D is obtained by adding diluted 5-20% autologous plateletconcentrate composition according to the invention to the culturemedium: cells are primed with the autologous platelet concentratecomposition (0.2 ml per well) just to cover base. Cells grow then as a3D fibrin gel matrix. Cells then differentiate to form biologicalscaffold or network in fibrin gel. Cell number is measured by dailycounting under a grid and to assess apoptosis: use inverted microscope(Olympus®).

After 3 to 6 weeks of incubation, the cells are harvested from thefibrin gel. Cell viability is assayed with classical Trypan blue methodand with bacteriological evaluation, including virus contamination. Theexpanded fibroblast cell extract obtained above is placed in a syringein presence of autologous platelet concentrate composition according tothe invention and the preparation is injected into face wrinkles, morespecifically under the wrinkles. Injections must be performed over thewhole face to cover forehead, jowls, molar region, cheeks, chin andneck. Cell expansion may be increased by photo light exposure of cellculture at 633 nm. The fibroblast cell preparation according to theinvention is useful for facial rejuvenation, amelioration of facialwrinkles and rhytids, treatment of skins damaged by radiation(radiodermatitis or sun damaged skin), aged skins or burned skins and/orin the amelioration of facial wrinkles, rhytids, acne (especially afterdermabrasion treatment), burns, rubella or small pox scars, vitiligo,lipoatrophy or lypodystrophy, such as AIDS-related lypodystrophy;Kaposi's sarcoma, skin keloids or Dupuytren's palmar fibromatosis and/orin skin rejuvenation treatments.

Example 5: Autologous Adipose Tissue & Fat Cell Association Preparation

Adipose tissue is aspirated preferably in the lower abdomen area,purified by PBS washing, centrifuged, with sedimentation in order toisolate the fat tissue from the triglyceride and debris. In themeantime, blood is collected and centrifuged for the preparation of aplasma concentrate or PRP composition according to the invention. Thecombination of the fat tissue and PRP composition is performed in thesyringe used for the fat aspiration once the triglycerides havediminished, by using a sterile transfer device for aspiration of PRPcomposition. The ratio of plasma concentrate and fat tissue is 10:10 forthe face and 10:3 for the breast and contouring.

Preferably, the injection is done extemporaneously, in a minimal periodof preparation time of both concentrated cells, and for a minimum timeof cell manipulation ex-vivo.

Examples of autologous cell association according to the invention canbe prepared by using the process according to the invention wherein forexample adipose stem cells or mesenchymal stem cells (MSCs) are providedunder step (d) or (e). MSCs may by isolated by collagenase(preadipocytes, pre-endothelial cells) and put in suspension with PRPand the preparation injected including subcutaneously, intra-articular,or for orthopedic surgery. The preparation may be combined with a soakacellular matrix to be applied directly on a wound, or may be cultivatedin laboratory before application. Adult adipose stem cells are isolatedby standard culture method in 5-20% vol. of an autologous plateletconcentrate composition according to the invention. The preparation isthen injected with an applicator into patients suffering from tissuedeficiencies, such as post traumatic deficiencies or age-relateddeficiencies for patients being around about 40 years-old. The fat cellpreparation according to the invention is useful for the treatment oflipoatrophy such as in HIV/AIDS patients and others with congenitalhemiatrophy of the face.

Example 6: Autologous Chondrocyte Cell Association Preparation

Examples of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinchondrocyte cells are provided under step (d) or (e).

Cartilage is isolated from the donor's knee (biopsy size 10×5 mm) anddiced. The cartilage chondrocyte cells are cultured for 4-6 weeks inmedium enriched with a diluted 5-20% autologous platelet concentratecomposition according to the invention. Cartilage cells are thenreleased by enzymatic digestion (collagenase and pronase). The cellpreparation is then incorporated surgically into the patient with deepchondral defects and damage.

The chondrocyte cell preparation according to the invention is usefulfor the treatment of deep cartilage damage and erosion or arthroscopy.

Another example of the use of a chondrocyte cell preparation accordingto the invention is the use in rhinoplasty without surgery by a singleinjection procedure: A patient suffering from congenital cartilage noseatrophy.

The day before injection, a biopsy of the cartilage of the ear 0.4*0.4cm is performed and placed in a sterile recipient filled with DMEM andantibiotic. The biopsy is treated with enzymatic digestion includingtrypsin and collagenase. The released chondrocytes are then re-suspendedin the autologous platelet concentrate composition according to theinvention.

The patient receives first a local anesthesia, and nose disinfection.Then, the above chondrocyte preparation is injected on the cartilagesurface and/or periosteum membrane of the site requiring augmentation ofvolume or lift. In a second phase, autologous platelet concentratecomposition according to the invention is injected into the superficialpart of the nose skin, in order to biostimulate regeneration and therejuvenation of the skin. After one hour, the injection is achieved andthe patient could return home. An exceptional recovery of viable cellsis observed: the amount of chondrocyte cells and plasma cells recoveredand injected was about 10⁹ cells. The chondrocyte cell preparationaccording to the invention is therefore useful for the treatment ofnasal cartilage defects, without surgical procedure, but only byinjection.

Example 7: Autologous Umbilical Cord Stem Cell Association Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinumbilical cord stem cells are provided under step (d) or (e). Umbilicalcord stem cells are isolated and then cryo-preserved and used to treatblood disorders.

The umbilical cord stem cell preparation according to the invention isuseful for the treatment of haematological diseases (like Thalassaemia).

The process of injection is resuspension of stem cells in the plasmaconcentrate followed by reinjection.

Example 8: Autologous Tendon Cell Association Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention whereintendon cells are provided under step (d) or (e).

Tendon fibroblast cells are isolated according to standard procedures in5-20% vol. of autologous platelet concentrate composition according tothe invention. The tendon fibroblast cells are cultured for about 1 toabout 3 weeks in culture medium enriched with an autologous plateletconcentrate composition according to the invention. Before injection,the cells are resuspended in freshly processed plasma concentrate. Thecell preparation is then injected into the patient at the injury site(e.g., torn tendon, arthritic area). The injection can be guided byechography, for localisation of the damaged site, and better graft ofthe implanted solution.

The injection of the tendon fibroblast cell preparation may also beperformed next to rotator cuff in shoulder: first the rotator cuff tearis repaired arthroscopically, then the tendon fibroblast cellpreparation is injected via a long catheter onto the sutured area. Thisimproves the healing of the tendon fibroblast at the edge of the rotatorcuff, prevents haematoma in confined space under the acromion, andprevents frozen shoulder by speeding up healing and enhancingrehabilitation and joint movement. The tendon cell preparation accordingto the invention is useful for the treatment of torn tendons, arthritisin joint caused by traumas or by aging, rotator cuff in shoulder.

Example 9: Autologous Ligament and Gingival Cell Association Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinperiosteal membrane and gingival cells are provided under step (d) or(e).

Under general and local anesthesia, periosteum (approximately 10×10 mm)is aseptically harvested from the buccal side of the mandibular body infour healthy female beagle dogs. The harvested periosteum is cut into3×3 mm pieces. The tissues are placed directly on a 6-well plate andcultured (for about 3 to about ??? weeks) in a humidified atmosphere of5% CO2 and 95% air at 37° C. in a culture medium enriched with a 5-20%autologous platelet concentrate composition according to the invention.The periosteal membrane and gingival cells are isolated by enzymaticdigestion and cultured by static technique.

Typically, 6 weeks of culture is sufficient to obtain appropriateperiosteal membrane thickness for grafting.

The autologous platelet concentrate composition and the cell preparationare injected after resuspension into the patient at the injury site.

Example 10: Autologous Corneal Cell Association Preparation

Preparation on a petri dish with a thermo sensitive coating membrane,for the collection of cells without enzymatic solution like collagenaseor trypsin.

Examples of autologous cell association according to the invention canbe prepared by using the process according to the invention whereincorneal cells are provided under step (d) or (e).

A biopsy is taken from the epicanthis on the edge of the comea and thecorneal limbal stem cells were expanded for autologous transplantationin the same person after 4 weeks of culture in Petri dishes or flaskscoated with an autologous platelet concentrate composition according tothe invention.

The corneal cultured stem cells (of limbal origin) may be ex vivoengineered onto the surface of de-epithelialised human amnion in amonolayer, after seeding the construct with a suspension of cultured andviable corneal keratinocytes according to the invention. About 500,000cells are used for the seeding and the cells are allowed to cover thesurface of the construct after further incubation of about another 3weeks. The engineering with cells occurs after about three weeks ofprimary cell culture, and re-seeding may be necessary. The resultingbiological cell-biocomposite construct consists of collagen, amnionfibers and corneal keratinocytes, consisting of membrane and monolayerof cells. The corneal cell preparation according to the invention can bespread onto a dissolvable contact lens that is applied to the damagedcomea. The contact lens disappears and the cells close the cornealdefect. The corneal cell preparation according to the invention can beadministered topically in eye drops in patients suffering from dry eyesymptoms. Alternatively, the above amnion can be used on its own on thescarred comea or the construct and the cell preparation according to theinvention can be attached to the inside of a biological or artificialcontact lens and then applied to the comea and covered with an eye pad.

Alternatively, corneal cells are resuspended in freshly processed plasmaconcentrate before application.

The corneal cell preparation according to the invention is useful inalleviating the pain of dry eye, for the treatment of Stevens-JohnsonSyndrome and corneal blindness due to acid and corrosive alkali burns inindustry, corneal ulcers such as recalcitrant neurotrophic, herpetic andimmunologically induced corneal ulceration.

Example 11: Autologous Bone Marrow Cell Association Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention wherein bonemarrow cells are provided under step (d) or (e).

Hip bone marrow is aspirated, harvested and centrifuged in aready-to-use device for the preparation of a bone marrow concentrate inorder to separate red blood cells.

The bone marrow cell preparation is used alone or then admixed to theplatelet concentrate according to the invention or centrifuged againwith calcium gluconate to form a suturable membrane and applied orinjected with an applicator to the injured site of the patients. Thebone marrow cell preparation according to the invention is useful forthe treatment of bone defect or cartilage defect. The bone marrow cellpreparation may be used alone or in combination with a plasmaconcentrate according to the invention. A cartilage membrane may be usedas well with calcium gluconate.

Example 12: Autologous Schwann Cell Association Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinSchwann cells are provided under step (d) or (e).

Under local anaesthesia, a biopsy is performed on either the N.Saphenous or N. Suralis in the lower extremity. The nerve biopsy is cutinto small blocks and primary cultures are induced in Petri dishesenriched with an autologous platelet concentrate composition accordingto the invention.

Monolayers are expanded in 3D and the cells are eventually harvested bytrypsin digestion and resuspended in a freshly harvested plateletconcentrate in a syringe for local infiltration of the surgicallyexposed and damaged spinal cord. The cultivated cells have been shown tocontain myelin. The Schwann cell preparation according to the inventionis useful for the treatment of peripheral nerve damage, nerve suture andspinal cord injury.

Example 13: Autologous Human Islet Cell Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinpancreas islet cells are provided under step (d) or (e).

Pancreas islets are harvested by open biopsy and separated byconventional enzymatic digestion and Ficol or Hypaqe separation (Page etal., 2007, Diba. Vas. Dis. Res., 7-12) then resuspended in a mediumenriched with an autologous platelet concentrate composition accordingto the invention.

The pancreas islet cell preparation is then injected via the portal veininto the liver.

The pancreas islet cell preparation according to the invention is usefulfor the treatment of type 1 diabetes or insulin-dependent diabetes andfor the reversal of hyperglycaemia of diabetes mellitus.

Example 14: Autologous Human Osteoblast Cell Preparation

An example of autologous cell association according to the invention canbe prepared by using the process according to the invention whereinosteoblast cells are provided under step (d) or (e).

Cortical punch bone biopsy is derived from the iliac crest or equivalentsite (maxilla) under local anaesthesia. The bone biopsy is placedaseptically in DMEM medium at 4° C., or equivalent transport medium bythose experienced in the art of bone and osteoblast culture ex vivo. Thebone biopsy is then diced and digested in diluted 10% type-1 collagenase(Sigma or Boehringer) at 37° C. for 15 min under laminar flow hood.Alternatively, trypsin digestion (Worthington) may be used. Enzymaticdigestion is terminated with three washes with an autologous plateletconcentrate composition according to the invention, at 10% in DMEM at 4°C. The preparation is centrifuged, pelleted and resuspended. The bonefragments are plated on Petri dishes or flasks as explants withair-lifting technology in an autologous platelet concentrate compositionaccording to the invention. The preparation is cultured at 37° C. withantibiotics, gentimicin and amphotericin-B under a gas flow of 95% airand 5% CO2. The culture medium is changed three times per week, eachtime spiking DMEM medium with 10-20% vol. of an autologous plateletconcentrate composition according to the invention. The cell viabilityand morphology are evaluated three times a week to assess cell crawling,apoptosis and 3D dimensional monolayer progression. The formation ofmicrofilament and differentiation is assessed by inverted microscopy(Olympus®). Absence of bacterial and viral contamination is checked.Osteoblasts can be engineered in a freshly harvested autologous fibrinpolymer membrane prepared by centrifugation of platelet concentrate withcalcium gluconate at a ratio of about 10:3 or about 10:10. The fibrinpolymer is suturing. Osteoblasts can be engineered onto human amnion tocreate cell biocomposite scaffold and cell monolayer carrier/constructafter membrane seeding with 100,000 cells as obtained above and allowingmonolayer membrane expansion over 3-4 weeks allowing unique constructionof osteoblast-amnion-membrane construct for use and transfer to cover abone defect or grafted area following non-union of fracture in any site.Osteoblasts can alternatively be mixed to a fresh bone marrowconcentrate and platelet concentrate before injection/application, andfurther combined with autologous thrombin serum when there is a need ofa soft gel.

The osteoblast cell preparation according to the invention is useful forthe treatment of bone defects, bone grafts or bone disorders.

1. A separator system for the preparation of a thrombin serum,comprising: a tube; a thixotropic gel disposed in the tube, thethixotropic gel adapted for separating thrombin serum; the tube adaptedto be centrifuged for a sufficient length of time until liberation ofthrombin serum.
 2. The separator system according to claim 1, whereincentrifugation is performed at a force between about 1000 g and up toabout 2000 g for a time selected from about 20 minutes up to about 40minutes or at a force of about 1500 g for a time selected from about 25minutes up to about 35 minutes or at a force of about 1500 g for about30 minutes.
 3. The separator system according to claim 1, wherein saidtube only contains a thixotropic gel.
 4. The separator system accordingto claim 1, wherein said tube is a glass tube.
 5. A thrombin serumproduced by centrifuging whole blood in the separator system of claim 1.6. A separator system for the preparation of a thrombin serum,comprising: a tube suitable for centrifugation; a thixotropic geldisposed in the tube, the thixotropic gel adapted for separatingthrombin serum; and a coagulation activator disposed in the tube.
 7. Thesystem of claim 6, wherein the tube only contains the thixotropic geldisposed below the coagulation activator.
 8. A kit comprising: aseparator system according to claim 1; and phlebotomy accessories. 9.The kit of claim 8, wherein the kit comprises: a double syringe forsimultaneous dispensation of a thrombin serum and a plateletconcentrate.
 10. The kit of claim 8, further comprising a centrifuge forcentrifuging the tube.
 11. A method for the preparation of a thrombinserum comprising centrifuging a separator system according to claim 1until the formation of thrombin serum.
 12. A method for the preparationof a thrombin serum comprising centrifuging a separator system accordingto claim 6 until the formation of thrombin serum.
 13. A method oftreating a patient with platelet rich plasma and thrombin serum,comprising: a) admixing a platelet rich plasma with the thrombin serumof claim 5; and b) administering the platelet rich plasma and thrombinserum to the patient.
 14. The method of claim 13, wherein the treatingcomprises tissue healing or regeneration treatments, traumatic orsurgical wound treatments, vasculitis treatments, ulcer or decubitussore treatments, radiodermatitis treatments, or treatments for closingfistulas.
 15. The method of claim 13, wherein the treating comprisesskin damage or skin rejuvenation, facial wrinkle treatments, rhytidstreatments, acne treatments, burn treatments, rubella or small pox scartreatments, vitiligo treatments, lipoatrophy or lipodystrophytreatments, Kaposi's sarcoma treatments, skin keloid treatments, orDupuytren's palmar fibromatosis treatments.
 16. The method of claim 13,wherein the treating comprises bone, cartilage and articular disordertreatments, chondral damage treatments, arthritis treatments, cartilagetreatments, torn tendon treatments, or rotator cuff treatments.
 17. Themethod of claim 13, wherein the treating comprises cosmetic, anti-aging,or skin repair treatments, scar repair treatments, wrinkle fillingtreatments, lipoatrophy treatments, autogenous rejuvenation, or fatdeficiency treatments, alone on in combination with at least oneanti-aging agent.
 18. A method for the preparation of a wound or tissuehealing composition, comprising the steps of: a) Providing a thrombinserum according to claim 5, and b) Admixing the thrombin serum with aplatelet-rich plasma (PRP) composition or a platelet concentratecomposition
 19. The method for the preparation of a wound or tissuehealing composition according to claim 18, wherein said composition isfurther admixed with at least one cell extract, cell composition,tricalcium phosphate (TCP), bone substitute, chitosan, hyaluronic acid,cream, cream mask, fat cells, fat tissue, bone marrow concentrate,lubricin, cd-gelatin, botulinum toxin, calcium gluconate and/or stemcells, and/or wherein said cell extract is selected from an extract ofkeratinocytes, bone marrow cells, osteoblasts, chondrocytes,fibroblasts, periosteum or corneal cells, melanocytes, Langerhans cells,fat cells, muscle cells, umbilical cord cells, mesenchymal stem cells(MSCs), preadipocytes, adipocytes, stem cells, periosteal cells,gingival cells, pre-endothelial cells, Schwann cells, ligament cells,tendon cells or pancreas islet cells, and/or wherein said whole blood,thrombin serum, platelet-rich plasma, platelet concentrate, cellcomposition, stem cells, fat cells, fat tissue, bone marrow concentrateand/or the cell extract is autologous, and/or wherein said platelet richplasma or platelet concentrate is admixed in an about 1:1 ratio, or anabout 3:1 ratio or an about 10:1 ratio to said thrombin serum, and/orwherein said thrombin serum and said platelet rich plasma or plateletconcentrate are prepared simultaneously, and/or wherein said thrombinserum and said platelet rich plasma or platelet concentrate areprepared, respectively, by centrifugation for about 8 minutes to about10 minutes, and/or wherein said thrombin serum and said platelet richplasma or platelet concentrate are prepared at the same time byindividual centrifugation for about 8 minutes to about 10 minutes,and/or wherein said thrombin serum and said platelet rich plasma orplatelet concentrate are applied simultaneously.
 20. A wound healant ortissue healant composition, comprising: a) a thrombin serum according toclaim 5, and b) a platelet-rich plasma (PRP) composition or plasmaconcentrate composition.
 21. A wound healant composition or tissuehealant composition according to claim 20, further comprising a)hyaluronic acid, and/or b) at least one cell extract, cell composition,tricalcium phosphate (TCP), bone substitute, chitosan, hyaluronic acid,cream, cream mask, fat cells, fat tissue, bone marrow concentrate,lubricin, cd-gelatin, botulinum toxin, calcium gluconate and/or stemcells, and/or c) a platelet-rich plasma, platelet concentrate, thrombinserum, cell composition, stem cells, fat cells, fat tissue, bone marrowconcentrate and/or the cell extract, wherein said cell extract isselected from an extract of keratinocytes, bone marrow cells,osteoblasts, chondrocytes, fibroblasts, periosteum or corneal cells,melanocytes, Langerhans cells, fat cells, muscle cells, umbilical cordcells, mesenchymal stem cells (MSCs), preadipocytes, adipocytes, stemcells, periosteal cells, gingival cells, pre-endothelial cells, Schwanncells, ligament cells, tendon cells or pancreas islet cells, and/orwherein said platelet-rich plasma, plasma concentrate, thrombin serum,cell composition, stem cells, fat cells, fat tissue, bone marrowconcentrate and/or the cell extract is autologous.